U.S. patent application number 16/496135 was filed with the patent office on 2020-02-06 for rna bacterial vaccines.
This patent application is currently assigned to ModernaTX, Inc.. The applicant listed for this patent is ModernaTX, Inc.. Invention is credited to Giuseppe Ciaramella, Nadia Cohen, Elisabeth Narayanan.
Application Number | 20200038499 16/496135 |
Document ID | / |
Family ID | 63586591 |
Filed Date | 2020-02-06 |
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United States Patent
Application |
20200038499 |
Kind Code |
A1 |
Narayanan; Elisabeth ; et
al. |
February 6, 2020 |
RNA BACTERIAL VACCINES
Abstract
The disclosure relates to (i) a bacterial vaccine, comprising:
at least one RNA polynucleotide having an open reading frame
encoding at least one mutated bacterial antigenic polypeptide,
wherein the mutated bacterial antigenic polypeptide comprises at
least one asparagine (Asn) amino acid substitution; and (ii) a
Streptococcal vaccine, comprising: at least one RNA polynucleotide
having an open reading frame encoding at least one Streptococcal
antigenic polypeptide, such as pneumolysin. Incorporating the RNA
in a cationic lipid nanoparticle and a method of inducing an immune
response with said vaccine are also disclosed.
Inventors: |
Narayanan; Elisabeth;
(Cambridge, MA) ; Cohen; Nadia; (Cambridge,
MA) ; Ciaramella; Giuseppe; (Sudbury, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ModernaTX, Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
ModernaTX, Inc.
Cambridge
MA
|
Family ID: |
63586591 |
Appl. No.: |
16/496135 |
Filed: |
March 22, 2018 |
PCT Filed: |
March 22, 2018 |
PCT NO: |
PCT/US2018/023850 |
371 Date: |
September 20, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62474811 |
Mar 22, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/09 20130101;
A61K 2039/55555 20130101; A61K 39/092 20130101; A61K 31/7105
20130101; A61K 31/7115 20130101; A61K 2039/53 20130101; A61P 31/04
20180101; A61K 2039/54 20130101 |
International
Class: |
A61K 39/09 20060101
A61K039/09 |
Claims
1. A bacterial vaccine, comprising: at least one RNA polynucleotide
having an open reading frame encoding at least one mutated
bacterial antigenic polypeptide, wherein the mutated bacterial
antigenic polypeptide comprises at least one asparagine (Asn) amino
acid of a corresponding wild type bacterial antigenic polypeptide
which has been replaced with a non-Asn amino acid.
2. The bacterial vaccine of claim 1, wherein the RNA polynucleotide
is formulated in a cationic lipid nanoparticle.
3. The bacterial vaccine of claim 1 or 2, wherein the mutated
bacterial antigenic polypeptide has one Asn amino acid of a
corresponding wild type bacterial antigenic polypeptide which has
been replaced with a non-Asn amino acid.
4. The bacterial vaccine of claim 1 or 2, wherein the mutated
bacterial antigenic polypeptide has two Asn amino acids of a
corresponding wild type bacterial antigenic polypeptide which have
been replaced with a non-Asn amino acid.
5. The bacterial vaccine of claim 1 or 2, wherein the mutated
bacterial antigenic polypeptide has three Asn amino acids of a
corresponding wild type bacterial antigenic polypeptide which have
been replaced with a non-Asn amino acid.
6. The bacterial vaccine of claim 1 or 2, wherein the mutated
bacterial antigenic polypeptide has four Asn amino acids of a
corresponding wild type bacterial antigenic polypeptide which have
been replaced with a non-Asn amino acid.
7. The bacterial vaccine of claim 1 or 2, wherein the mutated
bacterial antigenic polypeptide has five Asn amino acids of a
corresponding wild type bacterial antigenic polypeptide which have
been replaced with a non-Asn amino acid.
8. The bacterial vaccine of any one of claims 1-7, wherein the Asn
amino acid has been replaced with a Ala amino acid.
9. The bacterial vaccine of any one of claims 1-8, wherein the
mutated bacterial antigenic polypeptide has greater than 80%
sequence identity to a wild type bacterial antigenic
polypeptide.
10. The bacterial vaccine of any one of claims 1-8, wherein the
mutated bacterial antigenic polypeptide has greater than 90%
sequence identity to a wild type bacterial antigenic
polypeptide.
11. The bacterial vaccine of any one of claims 1-8, wherein the
mutated bacterial antigenic polypeptide has greater than 95%
sequence identity to a wild type bacterial antigenic
polypeptide.
12. The bacterial vaccine of any one of claims 1-8, wherein the
mutated bacterial antigenic polypeptide has greater than 98%
sequence identity to a wild type bacterial antigenic
polypeptide.
13. The bacterial vaccine of any one of claims 1-12, wherein the
bacterial vaccine produces a lower IgG titer than an RNA vaccine
encoding a corresponding wild type antigen.
14. The bacterial vaccine of any one of claims 1-13, wherein the
bacterial vaccine has enhanced neutralization activity relative to
an RNA vaccine encoding a corresponding wild type antigen.
15. The bacterial vaccine of any one of claims 1-14, wherein the
mutated bacterial antigenic polypeptide is a mutated antigen of an
infectious bacteria selected from the group consisting of
Streptococcus and Staphylococcus.
16. The bacterial vaccine of claim 15, wherein the Streptococcus is
Streptococcus pneumoniae.
17. The bacterial vaccine of claim 15 or 16, wherein the mutated
antigen is a pneumolysin.
18. A method of vaccinating a subject, comprising administering the
bacterial vaccine of any one of claims 1-17 to the subject in an
effective amount to induce an immune response against the bacteria
in the subject.
19. The method of claim 17, wherein the immune response is an
enhanced neutralization activity relative to an RNA vaccine
encoding a corresponding wild type antigen.
20. A Streptococcal vaccine, comprising: at least one RNA
polynucleotide having an open reading frame encoding at least one
Streptococcal antigenic polypeptide.
21. The Streptococcal vaccine of claim 20, wherein the
Streptococcal antigenic polypeptide is a Streptococcus pneumoniae
antigenic polypeptide.
22. The Streptococcal vaccine of claim 20 or 21, wherein the
Streptococcal antigenic polypeptide is a pneumolysin.
23. The Streptococcal vaccine of claim 22, wherein the pneumolysin
has a wild type pneumolysin sequence.
24. The Streptococcal vaccine of claim 22, wherein the pneumolysin
has a modified pneumolysin sequence.
25. The Streptococcal vaccine of claim 24, wherein the modified
pneumolysin sequence includes a D205R mutation.
26. The Streptococcal vaccine of any one of claims 20-25, wherein
the at least one RNA polynucleotide has a nucleic acid sequence
that has at least 80% identity to any one of SEQ ID NO: 6-8, but
does not include wild-type mRNA sequence.
27. The Streptococcal vaccine of any one of claims 20-25, wherein
the at least one RNA polynucleotide has a nucleic acid sequence
that has at least 85% identity to any one of SEQ ID NO: 6-8, but
does not include wild-type mRNA sequence.
28. The Streptococcal vaccine of any one of claims 20-25, wherein
the at least one RNA polynucleotide has a nucleic acid sequence
that has at least 90% identity to any one of SEQ ID NO: 6-8, but
does not include wild-type mRNA sequence.
29. The Streptococcal vaccine of any one of claims 20-25, wherein
the at least one RNA polynucleotide has a nucleic acid sequence
that has at least 95% identity to any one of SEQ ID NO: 6-8, but
does not include wild-type mRNA sequence.
30. The Streptococcal vaccine of any one of claims 20-25, wherein
the at least one RNA polynucleotide has a nucleic acid sequence
that has at least 98% identity to any one of SEQ ID NO: 6-8, but
does not include wild-type mRNA sequence.
31. The Streptococcal vaccine of any one of claims 20-30, wherein
the Streptococcal antigenic polypeptide has an amino acid sequence
that has at least 90% identity to an amino acid sequence identified
by any one of SEQ ID NO: 10-29, but does not include wild-type
protein sequence.
32. The Streptococcal vaccine of any one of claims 20-30, wherein
the Streptococcal antigenic polypeptide has an amino acid sequence
that has at least 95% identity to an amino acid sequence identified
by any one of SEQ ID NO: 10-29, but does not include wild-type
protein sequence.
33. The Streptococcal vaccine of any one of claims 20-30, wherein
the Streptococcal antigenic polypeptide has an amino acid sequence
that has at least 99% identity to an amino acid sequence identified
by any one of SEQ ID NO: 10-29, but does not include wild-type
protein sequence.
34. The Streptococcal vaccine of any one of claims 20-30, wherein
the Streptococcal antigenic polypeptide has an amino acid sequence
of any one of SEQ ID NO: 10-29.
35. The Streptococcal vaccine of any one of claims 20-25, wherein
the at least one RNA polynucleotide has a nucleic acid sequence of
any one of SEQ ID NO: 6-8.
36. The Streptococcal vaccine of any one of claims 20-35, wherein
the RNA polynucleotide is formulated in a cationic lipid
nanoparticle.
37. The bacterial or Streptococcal vaccine of any one of claims
1-17 and 20-36, wherein the at least one RNA polynucleotide
comprises at least one chemical modification.
38. The bacterial or Streptococcal vaccine of claim 37, wherein the
chemical modification is selected from pseudouridine,
N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine,
4'-thiouridine, 5-methylcytosine, 5-methyluridine,
2-thio-1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,
2-thio-dihydropseudouridine, 2-thio-dihydrouridine,
2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,
4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,
4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,
5-methoxyuridine and 2'-O-methyl uridine.
39. The bacterial or Streptococcal vaccine of claim 37 or 38,
wherein the chemical modification is in the 5-position of the
uracil.
40. The bacterial or Streptococcal vaccine of claim 37 or 38,
wherein the chemical modification is a N1-methylpseudouridine or
N1-ethylpseudouridine.
41. The bacterial or Streptococcal vaccine of claim 37 or 38,
wherein at least 80% of the uracil in the open reading frame have a
chemical modification.
42. The bacterial or Streptococcal vaccine of claim 37 or 38,
wherein at least 90% of the uracil in the open reading frame have a
chemical modification.
43. The bacterial or Streptococcal vaccine of claim 37 or 38,
wherein 100% of the uracil in the open reading frame have a
chemical modification.
44. The bacterial or Streptococcal vaccine of claim 37 or 38,
wherein 100% of the uracil in the open reading frame is modified to
include N1-methyl pseudouridine at the 5-position of the
uracil.
45. The bacterial or Streptococcal vaccine of any one of claims
1-17 and 20-36, wherein at least one RNA polynucleotide further
encodes at least one 5' terminal cap.
46. The bacterial or Streptococcal vaccine of claim 45, wherein the
5' terminal cap is 7mG(5')ppp(5')NlmpNp.
47. The bacterial or Streptococcal vaccine of any one of claims
2-17 and 36-46, wherein the cationic lipid nanoparticle has a mean
diameter of 50-200 nm.
48. The bacterial or Streptococcal vaccine of any one of claims
2-17 and 36-46, wherein the cationic lipid nanoparticle comprises a
cationic lipid, a PEG-modified lipid, a sterol and a non-cationic
lipid.
49. The bacterial or Streptococcal vaccine of any one of claims
2-17 and 36-46, wherein the cationic lipid nanoparticle comprises a
molar ratio of about 20-60% cationic lipid, 0.5-15% PEG-modified
lipid, 25-55% sterol, and 5-25% non-cationic lipid.
50. The bacterial or Streptococcal vaccine of claim 48 or 49,
wherein the cationic lipid is an ionizable cationic lipid and the
non-cationic lipid is a neutral lipid, and the sterol is a
cholesterol.
51. The bacterial or Streptococcal vaccine of claim 49 or 50,
wherein the cationic lipid is selected from
2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).
52. The bacterial or Streptococcal vaccine of any one of claims
47-51, wherein the cationic lipid nanoparticle comprises a compound
of Formula (I), optionally Compound 3, 18, 20, 25, 26, 29, 30, 60,
108-112, or 122.
53. The bacterial or Streptococcal vaccine of any one of claims
47-51, wherein the cationic lipid nanoparticle comprises a compound
of Formula (II).
54. The bacterial or Streptococcal vaccine of any one of claims
47-51, wherein the cationic lipid nanoparticle has a polydispersity
value of less than 0.4.
55. The bacterial or Streptococcal vaccine of any one of claims
47-51, wherein the cationic lipid nanoparticle has a net neutral
charge at a neutral pH value.
56. The bacterial or Streptococcal vaccine of any one of claims
1-17 and 20-55, further comprising an adjuvant.
57. The bacterial or Streptococcal vaccine of any one of claims
1-17 and 20-56, wherein, wherein the open reading frame is
codon-optimized.
58. The bacterial or Streptococcal vaccine of any one of claims
1-17 and 20-57, wherein the vaccine is multivalent.
59. The bacterial or Streptococcal vaccine of any one of claims
1-17 and 20-58, formulated in an effective amount to produce an
antigen-specific immune response.
60. The bacterial or Streptococcal vaccine of any one of claims
1-17 and 20-58 for use in a method of inducing an antigen specific
immune response in a subject, the method comprising administering
to the subject the vaccine in an amount effective to produce an
antigen specific immune response in the subject.
61. A pharmaceutical composition for use in vaccination of a
subject comprising an effective dose of a bacterial or
Streptococcal vaccine of any one of claims 1-17 and 20-58, wherein
the effective dose is sufficient to produce detectable levels of
antigen as measured in serum of the subject at 1-72 hours post
administration.
62. The composition of claim 61, wherein the cut off index of the
antigen is 1-2.
63. A pharmaceutical composition for use in vaccination of a
subject comprising an effective dose of a bacterial or
Streptococcal vaccine of any one of claims 1-17 and 20-58, wherein
the effective dose is sufficient to produce a 1,000-10,000
neutralization titer produced by neutralizing antibody against said
antigen as measured in serum of the subject at 1-72 hours post
administration.
64. A composition comprising a bacterial or Streptococcal vaccine
of any one of claims 1-17 and 20-58 formulated in a lipid
nanoparticle comprising compounds of Formula (I): ##STR00005## or a
salt or isomer thereof, wherein: R.sub.1 is selected from the group
consisting of C.sub.5-30 alkyl, C.sub.5-20 alkenyl, --R*YR'',
--YR'', and --R''M'R'; R.sub.2 and R.sub.3 are independently
selected from the group consisting of H, C.sub.1-14 alkyl,
C.sub.2-14 alkenyl, --R*YR'', --YR'', and --R*OR'', or R.sub.2 and
R.sub.3, together with the atom to which they are attached, form a
heterocycle or carbocycle; R.sub.4 is selected from the group
consisting of a C.sub.3-6 carbocycle, --(CH.sub.2).sub.nQ,
--(CH.sub.2).sub.nCHQR, --CHQR, --CQ(R).sub.2, and unsubstituted
C.sub.1-6 alkyl, where Q is selected from a carbocycle,
heterocycle, --OR, --O(CH.sub.2)--N(R).sub.2, --C(O)OR, --OC(O)R,
--CX.sub.3, --CX.sub.2H, --CXH.sub.2, --CN, --N(R).sub.2,
--C(O)N(R).sub.2, --N(R)C(O)R, --N(R)S(O).sub.2R,
--N(R)C(O)N(R).sub.2, --N(R)C(S)N(R).sub.2, --N(R)R.sub.8,
--O(CH.sub.2).sub.nOR, --N(R)C(.dbd.NR.sub.9)N(R).sub.2,
--N(R)C(.dbd.CHR.sub.9)N(R).sub.2, --OC(O)N(R).sub.2, --N(R)C(O)OR,
--N(OR)C(O)R, --N(OR)S(O).sub.2R, --N(OR)C(O)OR,
--N(OR)C(O)N(R).sub.2, --N(OR)C(S)N(R).sub.2,
--N(OR)C(.dbd.NR.sub.9)N(R).sub.2,
--N(OR)C(.dbd.CHR.sub.9)N(R).sub.2, --C(.dbd.NR.sub.9)N(R).sub.2,
--C(.dbd.NR.sub.9)R, --C(O)N(R)OR, and --C(R)N(R).sub.2C(O)OR, and
each n is independently selected from 1, 2, 3, 4, and 5; each
R.sub.5 is independently selected from the group consisting of
C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; each R.sub.6 is
independently selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H; M and M' are independently
selected from --C(O)O--, --OC(O)--, --C(O)N(R')--, --N(R')C(O)--,
--C(O)--, --C(S)--, --C(S)S--, --SC(S)--, --CH(OH)--,
--P(O)(OR')O--, --S(O).sub.2--, --S--S--, an aryl group, and a
heteroaryl group; R.sub.7 is selected from the group consisting of
C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; R.sub.8 is selected from
the group consisting of C.sub.3-6 carbocycle and heterocycle;
R.sub.9 is selected from the group consisting of H, CN, NO.sub.2,
C.sub.1-6 alkyl, --OR, --S(O).sub.2R, --S(O).sub.2N(R).sub.2,
C.sub.2-6 alkenyl, C.sub.3-6 carbocycle and heterocycle; each R is
independently selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H; each R' is independently selected
from the group consisting of C.sub.1-18 alkyl, C.sub.2-18 alkenyl,
--R*YR'', --YR'', and H; each R'' is independently selected from
the group consisting of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
each R* is independently selected from the group consisting of
C.sub.1-12 alkyl and C.sub.2-12 alkenyl; each Y is independently a
C.sub.3-6 carbocycle; each X is independently selected from the
group consisting of F, Cl, Br, and I; and m is selected from 5, 6,
7, 8, 9, 10, 11, 12, and 13.
65. The vaccine of claim 64, wherein a subset of compounds of
Formula (I) includes those in which when R.sub.4 is
--(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR, --CHQR, or
--CQ(R).sub.2, then (i) Q is not --N(R).sub.2 when n is 1, 2, 3, 4
or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n
is 1 or 2.
66. The vaccine of claim 64, wherein a subset of compounds of
Formula (I) includes those in which R.sub.1 is selected from the
group consisting of C.sub.5-30 alkyl, C.sub.5-20 alkenyl, --R*YR'',
--YR'', and --R''M'R'; R.sub.2 and R.sub.3 are independently
selected from the group consisting of H, C.sub.1-14 alkyl,
C.sub.2-14 alkenyl, --R*YR'', --YR'', and --R*OR'', or R.sub.2 and
R.sub.3, together with the atom to which they are attached, form a
heterocycle or carbocycle; R.sub.4 is selected from the group
consisting of a C.sub.3-6 carbocycle, --(CH.sub.2).sub.nQ,
--(CH.sub.2).sub.nCHQR, --CHQR, --CQ(R).sub.2, and unsubstituted
C.sub.1-6 alkyl, where Q is selected from a C.sub.3-6 carbocycle, a
5- to 14-membered heteroaryl having one or more heteroatoms
selected from N, O, and S, --OR, --O(CH.sub.2).sub.nN(R).sub.2,
--C(O)OR, --OC(O)R, --CX.sub.3, --CX.sub.2H, --CXH.sub.2, --CN,
--C(O)N(R).sub.2, --N(R)C(O)R, --N(R)S(O).sub.2R,
--N(R)C(O)N(R).sub.2, --N(R)C(S)N(R).sub.2, --CRN(R).sub.2C(O)OR,
--N(R)R.sub.8, --O(CH.sub.2).sub.nOR,
--N(R)C(.dbd.NR.sub.9)N(R).sub.2,
--N(R)C(.dbd.CHR.sub.9)N(R).sub.2, --OC(O)N(R).sub.2, --N(R)C(O)OR,
--N(OR)C(O)R, --N(OR)S(O).sub.2R, --N(OR)C(O)OR,
--N(OR)C(O)N(R).sub.2, --N(OR)C(S)N(R).sub.2,
--N(OR)C(.dbd.NR.sub.9)N(R).sub.2,
--N(OR)C(.dbd.CHR.sub.9)N(R).sub.2, --C(.dbd.NR.sub.9)N(R).sub.2,
--C(.dbd.NR.sub.9)R, --C(O)N(R)OR, and a 5- to 14-membered
heterocycloalkyl having one or more heteroatoms selected from N, O,
and S which is substituted with one or more substituents selected
from oxo (.dbd.O), OH, amino, mono- or di-alkylamino, and C.sub.1-3
alkyl, and each n is independently selected from 1, 2, 3, 4, and 5;
each R.sub.5 is independently selected from the group consisting of
C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; each R.sub.6 is
independently selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H; M and M' are independently
selected from --C(O)O--, --OC(O)--, --C(O)N(R')--, --N(R')C(O)--,
--C(O)--, --C(S)--, --C(S)S--, --SC(S)--, --CH(OH)--,
--P(O)(OR')O--, --S(O).sub.2--, --S--S--, an aryl group, and a
heteroaryl group; R.sub.7 is selected from the group consisting of
C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; R.sub.8 is selected from
the group consisting of C.sub.3-6 carbocycle and heterocycle;
R.sub.9 is selected from the group consisting of H, CN, NO.sub.2,
C.sub.1-6 alkyl, --OR, --S(O).sub.2R, --S(O).sub.2N(R).sub.2,
C.sub.2-6 alkenyl, C.sub.3-6 carbocycle and heterocycle; each R is
independently selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H; each R' is independently selected
from the group consisting of C.sub.1-18 alkyl, C.sub.2-18 alkenyl,
--R*YR'', --YR'', and H; each R'' is independently selected from
the group consisting of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
each R* is independently selected from the group consisting of
C.sub.1-12 alkyl and C.sub.2-12 alkenyl; each Y is independently a
C.sub.3-6 carbocycle; each X is independently selected from the
group consisting of F, Cl, Br, and I; and m is selected from 5, 6,
7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.
67. The vaccine of claim 64, wherein a subset of compounds of
Formula (I) includes those in which R.sub.1 is selected from the
group consisting of C.sub.5-30 alkyl, C.sub.5-20 alkenyl, --R*YR'',
--YR'', and --R''M'R'; R.sub.2 and R.sub.3 are independently
selected from the group consisting of H, C.sub.1-14 alkyl,
C.sub.2-14 alkenyl, --R*YR'', --YR'', and --R*OR'', or R.sub.2 and
R.sub.3, together with the atom to which they are attached, form a
heterocycle or carbocycle; R.sub.4 is selected from the group
consisting of a C.sub.3-6 carbocycle, --(CH.sub.2).sub.nQ,
--(CH.sub.2).sub.nCHQR, --CHQR, --CQ(R).sub.2, and unsubstituted
C.sub.1-6 alkyl, where Q is selected from a C.sub.3-6 carbocycle, a
5- to 14-membered heterocycle having one or more heteroatoms
selected from N, O, and S, --OR, --O(CH.sub.2)--N(R).sub.2,
--C(O)OR, --OC(O)R, --CX.sub.3, --CX.sub.2H, --CXH.sub.2, --CN,
--C(O)N(R).sub.2, --N(R)C(O)R, --N(R)S(O).sub.2R,
--N(R)C(O)N(R).sub.2, --N(R)C(S)N(R).sub.2, --CRN(R).sub.2C(O)OR,
--N(R)R.sub.8, --O(CH.sub.2).sub.nOR,
--N(R)C(.dbd.NR.sub.9)N(R).sub.2,
--N(R)C(.dbd.CHR.sub.9)N(R).sub.2, --OC(O)N(R).sub.2, --N(R)C(O)OR,
--N(OR)C(O)R, --N(OR)S(O).sub.2R, --N(OR)C(O)OR,
--N(OR)C(O)N(R).sub.2, --N(OR)C(S)N(R).sub.2,
--N(OR)C(.dbd.NR.sub.9)N(R).sub.2,
--N(OR)C(.dbd.CHR.sub.9)N(R).sub.2, --C(.dbd.NR.sub.9)R,
--C(O)N(R)OR, and --C(.dbd.NR.sub.9)N(R).sub.2, and each n is
independently selected from 1, 2, 3, 4, and 5; and when Q is a 5-
to 14-membered heterocycle and (i) R.sub.4 is --(CH.sub.2).sub.nQ
in which n is 1 or 2, or (ii) R.sub.4 is --(CH.sub.2).sub.nCHQR in
which n is 1, or (iii) R.sub.4 is --CHQR, and --CQ(R).sub.2, then Q
is either a 5- to 14-membered heteroaryl or 8- to 14-membered
heterocycloalkyl; each R.sub.5 is independently selected from the
group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; each
R.sub.6 is independently selected from the group consisting of
C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; M and M' are
independently selected from --C(O)O--, --OC(O)--, --C(O)N(R')--,
--N(R')C(O)--, --C(O)--, --C(S)--, --C(S)S--, --SC(S)--,
--CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--, --S--S--, an aryl
group, and a heteroaryl group; R.sub.7 is selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; R.sub.8 is
selected from the group consisting of C.sub.3-6 carbocycle and
heterocycle; R.sub.9 is selected from the group consisting of H,
CN, NO.sub.2, C.sub.1-6 alkyl, --OR, --S(O).sub.2R,
--S(O).sub.2N(R).sub.2, C.sub.2-6 alkenyl, C.sub.3-6 carbocycle and
heterocycle; each R is independently selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; each R' is
independently selected from the group consisting of C.sub.1-18
alkyl, C.sub.2-18 alkenyl, --R*YR'', --YR'', and H; each R'' is
independently selected from the group consisting of C.sub.3-14
alkyl and C.sub.3-14 alkenyl; each R* is independently selected
from the group consisting of C.sub.1-12 alkyl and C.sub.2-12
alkenyl; each Y is independently a C.sub.3-6 carbocycle; each X is
independently selected from the group consisting of F, Cl, Br, and
I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or
salts or isomers thereof.
68. The vaccine of claim 64, wherein subset of compounds of Formula
(I) includes those in which R.sub.1 is selected from the group
consisting of C.sub.5-30 alkyl, C.sub.5-20 alkenyl, --R*YR'',
--YR'', and --R''M'R'; R.sub.2 and R.sub.3 are independently
selected from the group consisting of H, C.sub.2-14 alkyl,
C.sub.2-14 alkenyl, --R*YR'', --YR'', and --R*OR'', or R.sub.2 and
R.sub.3, together with the atom to which they are attached, form a
heterocycle or carbocycle; R.sub.4 is --(CH.sub.2).sub.nQ or
--(CH.sub.2).sub.nCHQR, where Q is --N(R).sub.2, and n is selected
from 3, 4, and 5; each R.sub.5 is independently selected from the
group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; each
R.sub.6 is independently selected from the group consisting of
C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; M and M' are
independently selected from --C(O)O--, --OC(O)--, --C(O)N(R')--,
--N(R')C(O)--, --C(O)--, --C(S)--, --C(S)S--, --SC(S)--,
--CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--, --S--S--, an aryl
group, and a heteroaryl group; R.sub.7 is selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; each R is
independently selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H; each R' is independently selected
from the group consisting of C.sub.1-18 alkyl, C.sub.2-18 alkenyl,
--R*YR'', --YR'', and H; each R'' is independently selected from
the group consisting of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
each R* is independently selected from the group consisting of
C.sub.1-12 alkyl and C.sub.1-12 alkenyl; each Y is independently a
C.sub.3-6 carbocycle; each X is independently selected from the
group consisting of F, Cl, Br, and I; and m is selected from 5, 6,
7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.
69. The vaccine of claim 64, wherein a subset of compounds of
Formula (I) includes those in which R.sub.1 is selected from the
group consisting of C.sub.5-30 alkyl, C.sub.5-20 alkenyl, --R*YR'',
--YR'', and --R''M'R'; R.sub.2 and R.sub.3 are independently
selected from the group consisting of C.sub.1-14 alkyl, C.sub.2-14
alkenyl, --R*YR'', --YR'', and --R*OR'', or R.sub.2 and R.sub.3,
together with the atom to which they are attached, form a
heterocycle or carbocycle; R.sub.4 is selected from the group
consisting of --(CH.sub.2).sub.nQ, --(CH.sub.2).sub.nCHQR, --CHQR,
and --CQ(R).sub.2, where Q is --N(R).sub.2, and n is selected from
1, 2, 3, 4, and 5; each R.sub.5 is independently selected from the
group consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; each
R.sub.6 is independently selected from the group consisting of
C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; M and M' are
independently selected from --C(O)O--, --OC(O)--, --C(O)N(R')--,
--N(R')C(O)--, --C(O)--, --C(S)--, --C(S)S--, --SC(S)--,
--CH(OH)--, --P(O)(OR')O--, --S(O).sub.2--, --S--S--, an aryl
group, and a heteroaryl group; R.sub.7 is selected from the group
consisting of C.sub.1-3 alkyl, C.sub.2-3 alkenyl, and H; each R is
independently selected from the group consisting of C.sub.1-3
alkyl, C.sub.2-3 alkenyl, and H; each R' is independently selected
from the group consisting of C.sub.1-18 alkyl, C.sub.2-18 alkenyl,
--R*YR'', --YR'', and H; each R'' is independently selected from
the group consisting of C.sub.3-14 alkyl and C.sub.3-14 alkenyl;
each R* is independently selected from the group consisting of
C.sub.1-12 alkyl and C.sub.1-12 alkenyl; each Y is independently a
C.sub.3-6 carbocycle; each X is independently selected from the
group consisting of F, Cl, Br, and I; and m is selected from 5, 6,
7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.
70. The vaccine of claim 64, wherein a subset of compounds of
Formula (I) includes those of Formula (IA): ##STR00006## or a salt
or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and 5; m
is selected from 5, 6, 7, 8, and 9; M.sub.1 is a bond or M';
R.sub.4 is unsubstituted C.sub.1-3 alkyl, or --(CH.sub.2).sub.nQ,
in which Q is OH, --NHC(S)N(R).sub.2, --NHC(O)N(R).sub.2,
--N(R)C(O)R, --N(R)S(O).sub.2R, --N(R)R.sub.8,
--NHC(.dbd.NR.sub.9)N(R).sub.2, --NHC(.dbd.CHR.sub.9)N(R).sub.2,
--OC(O)N(R).sub.2, --N(R)C(O)OR, heteroaryl or heterocycloalkyl; M
and M' are independently selected from --C(O)O--, --OC(O)--,
--C(O)N(R')--, --P(O)(OR')O--, --S--S--, an aryl group, and a
heteroaryl group; and R.sub.2 and R.sub.3 are independently
selected from the group consisting of H, C.sub.1-14 alkyl, and
C.sub.2-14 alkenyl.
71. A method of inducing an immune response in a subject, the
method comprising administering to the subject the Streptococcal
vaccine of any one of claims 20-59 in an amount effective to
produce an antigen-specific immune response in the subject.
72. The method of claim 71, wherein the antigen specific immune
response comprises a T cell response or a B cell response.
73. The method of claim 71 or 72, wherein the subject is
administered a single dose of the vaccine.
74. The method of claim 71 or 72, wherein the subject is
administered a booster dose of the vaccine.
75. The method of any one of claims 71-74, wherein the vaccine is
administered to the subject by intradermal injection or
intramuscular injection.
76. The method of any one of claims 71-75, wherein an
anti-antigenic polypeptide antibody titer produced in the subject
is increased by at least 1 log relative to a control.
77. The method of any one of claims 71-76, wherein an
anti-antigenic polypeptide antibody titer produced in the subject
is increased by 1-3 log relative to a control.
78. The method of any one of claims 71-77, wherein the
anti-antigenic polypeptide antibody titer produced in the subject
is increased at least 2 times relative to a control.
79. The method of any one of claims 71-78, wherein the
anti-antigenic polypeptide antibody titer produced in the subject
is increased 2-10 times relative to a control.
80. The method of any one of claims 76-79, wherein the control is
an anti-antigenic polypeptide antibody titer produced in a subject
who has not been administered a vaccine against the bacteria.
81. The method of any one of claims 76-79, wherein the control is
an anti-antigenic polypeptide antibody titer produced in a subject
who has been administered a live attenuated vaccine or an
inactivated vaccine against the bacteria.
82. The method of any one of claims 76-79, wherein the control is
an anti-antigenic polypeptide antibody titer produced in a subject
who has been administered a recombinant protein vaccine or purified
protein vaccine against the bacteria.
83. The method of any one of claims 71-82, wherein the effective
amount is a dose equivalent to an at least 2-fold reduction in the
standard of care dose of a recombinant protein vaccine or a
purified protein vaccine against the bacteria, and wherein an
anti-antigenic polypeptide antibody titer produced in the subject
is equivalent to an anti-antigenic polypeptide antibody titer
produced in a control subject administered the standard of care
dose of a recombinant protein vaccine or a purified protein vaccine
against the bacteria, respectively.
84. The method of any one of claims 71-82, wherein the effective
amount is a dose equivalent to an at least 2-fold reduction in the
standard of care dose of a live attenuated vaccine or an
inactivated vaccine against the bacteria, and wherein an
anti-antigenic polypeptide antibody titer produced in the subject
is equivalent to an anti-antigenic polypeptide antibody titer
produced in a control subject administered the standard of care
dose of a live attenuated vaccine or an inactivated vaccine against
the bacteria, respectively.
85. The method of any one of claims 71-82, wherein the effective
amount is a dose equivalent to an at least 2-fold reduction in the
standard of care dose of a adjuvanted peptide vaccine against the
bacteria, and wherein an anti-antigenic polypeptide antibody titer
produced in the subject is equivalent to an anti-antigenic
polypeptide antibody titer produced in a control subject
administered the standard of care dose of an adjuvanted peptide
vaccine against the bacteria.
86. The method of any one of claims 71-85, wherein the effective
amount is a total dose of 50 .mu.g-1000 .mu.g.
87. The method of claim 86, wherein the effective amount is a dose
of 25 .mu.g, 100 .mu.g, 400 .mu.g, or 500 .mu.g administered to the
subject a total of two times.
88. The method of any one of claims 71-87, wherein the efficacy of
the vaccine against the bacteria is greater than 65%.
89. The method of any one of claims 71-88, wherein the vaccine
immunizes the subject against the bacteria for up to 2 years.
90. The method of any one of claims 71-89, wherein the vaccine
immunizes the subject against the bacteria for more than 2
years.
91. The method of any one of claims 71-90, wherein the subject has
an age of about 12 to about 50 years old.
92. The method of any one of claims 71-91, wherein the subject has
been exposed to the bacteria, wherein the subject is infected with
the bacteria, or wherein the subject is at risk of infection by the
bacteria.
93. The method of any one of claims 71-92, wherein the subject is
immunocompromised.
Description
RELATED APPLICATION
[0001] This application claims the benefit under 35 U.S.C. .sctn.
119(e) of U.S. provisional application No. 62/474,811, filed Mar.
22, 2017, which is incorporated by reference herein in its
entirety.
BACKGROUND
[0002] Streptococcus pneumoniae, a gram-positive,
catalase-negative, facultative anaerobic bacterium, causes a
variety of serious human diseases such as pneumonia, bronchitis,
bacterial meningitis, sepsis, otitis media (ear infections), and
corneal ulcers. The bacterium typically colonizes the respiratory
tract, sinuses, and nasal cavities in healthy carriers, but may
become pathogenic in immunosuppressed organisms. In severe cases,
pneumococcal diseases may cause hearing loss, brain damage, and
death.
[0003] Pneumolysin (PLY, AJS15225.1; M17717.1), a putative major
virulence factor of Streptococcus pneumoniae, is a 53 kDa
pore-forming toxin consisting of 471 amino acids. Marriott et al.,
Curr Mol Med. 8(6):497-509 (2008). The toxin is inhibited by
cholesterol, and at high levels (greater than 50 hemolytic units),
it is lytic to all cells with cholesterol in the membrane. At
lower, sublytic concentrations, pneumolysin can induce apoptosis,
activate the host complement, and induce proinflammatory reactions
in immune cells. Pneumolysin is generally located in the bacterial
cytoplasm, but does not have an N-terminal secretion signal
sequence, so it is released when the pneumococcus undergoes
autolysis with N-acetyl-muramoyl-1-alanine amidase (Lyt A). Hirst
et al., Clin Exp Immunol. 138(2): 195-201 (2004). The toxin, a
water-soluble monomer, recognizes mammalian cells via its
C-terminal domain (domain 4), and assembles into circular prepores
of approximately 30-50 monomers on the surface of cholesterol-rich
membranes. When bound, the monomers undergo conformational changes,
resulting in a PLY .beta.-barrel pore that causes lysis of the
target cell. Lawrence et al., Sci Rep. 5:14352 (2015).
[0004] Due to drug-resistant pneumococci and the complexity of
vaccines needed to cover a broad spectrum of strains, it is
necessary to find new vaccines with antigens that offer protection
in a strain-independent manner and that can effectively abolish the
virulence of S. pneumoniae.
SUMMARY
[0005] Provided herein are highly effective bacterial vaccines that
are useful for treating, prophylactically and therapeutically,
bacterial infection. The bacterial vaccines are ribonucleic acid
(RNA) vaccines that enable production of a bacterial protein in a
host from messenger RNA (mRNA)) such that the protein can safely
elicit a robust immune response. The RNA (e.g., mRNA) vaccines of
the present disclosure may be used to induce a balanced immune
response against bacterial infections, comprising both cellular and
humoral immunity.
[0006] The RNA (e.g., mRNA) vaccines may be utilized in various
settings depending on the prevalence of the infection or the degree
or level of unmet medical need. The RNA (e.g. mRNA) vaccines may be
utilized to treat and/or prevent a bacterial infection. The RNA
(e.g., mRNA) vaccines have superior properties in that they may
produce much larger antibody titers and produce responses earlier
than commercially available vaccines. While not wishing to be bound
by theory, it is believed that the RNA (e.g., mRNA) vaccines, as
mRNA polynucleotides, are better designed to produce the
appropriate protein conformation upon translation as the RNA
vaccines co-opt natural cellular machinery. Unlike traditional
vaccines, which are manufactured ex vivo and may trigger unwanted
cellular responses, RNA (e.g., mRNA) vaccines are presented to the
cellular system in a more native fashion. Additionally, the ability
to produce proteins in human hosts that are not glycosylated, and
thus more closely mimic naturally occurring bacterial proteins have
been found to produce more robust immune responses that are
effective in neutralizing the bacteria and preventing bacterial
infection.
[0007] In some aspects the invention is a bacterial vaccine,
comprising at least one RNA polynucleotide having an open reading
frame encoding at least one bacterial antigenic polypeptide which
comprises a mutated N-linked glycosylation site. In some
embodiments, the mutated bacterial antigenic polypeptide comprises
at least one asparagine (Asn) amino acid of a corresponding
wild-type bacterial antigenic polypeptide which has been replaced
with a non-Asn amino acid.
[0008] In some embodiments the mutated bacterial antigenic
polypeptide has one, two, three, four, or five Asn amino acids of a
corresponding wild type bacterial antigenic polypeptide which have
been replaced with a non-Asn amino acid. In other embodiments the
Asn amino acid has been replaced with an Ala amino acid. In some
embodiments the mutated bacterial antigenic polypeptide has greater
than 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to
a wild type bacterial antigenic polypeptide.
[0009] In some embodiments the bacterial vaccine produces a lower
IgG titer than an RNA vaccine encoding a corresponding wild type
antigen. In other embodiments the bacterial vaccine has enhanced
neutralization activity relative to an RNA vaccine encoding a
corresponding wild type antigen.
[0010] The mutated bacterial antigenic polypeptide in some
embodiments is a mutated antigen of an infectious bacteria selected
from the group consisting of Acetobacter, Acinetobacter,
Actinomyces, Agrobacterium, Anaplasma, Azorhizobia, Bacillus,
Bacteroides, Bartonella, Bordetella, Borrelia, Brucella,
Burkkolderia, Calymmatobacterium, Campylobacter, Chlamydia,
Chlamydophila, Clostridium, Corynebacterium, Coxiella, Ehrlichia,
Enterobacter, Enterococcus, Escherichia, Francisella,
Fusobacterium, Gardnerella, Haemophilus, Helicobacter, Klebsiella,
Lactobacillus, Legionella, Listeria, Methanobacterium,
Microbacterium, Micrococcus, Moraxella, Mycobacterium, Mycoplasma,
Neisseria, Pasteurella, Peptostreptococcus, Porphyromonas,
Prevotella, Pseudomonas, Rhizobium, Rickettsia, Rochalimaea,
Rothia, Salmonella, Shigella, Staphylococcus, Stenotrophomonas,
Streptococcus, Treponema, Vibrio, Walbachia, and Yersinia. In other
embodiments the Streptococcus is Streptococcus pneumoniae. In yet
other embodiments the mutated antigen is a pneumolysin.
[0011] In other aspects, the invention is a Streptococcal vaccine,
having at least one RNA polynucleotide having an open reading frame
encoding at least one Streptococcal antigenic polypeptide. In some
embodiments the Streptococcus is Streptococcus pneumoniae. In yet
other embodiments the mutated antigen is a pneumolysin. In some
embodiments the pneumolysin has a wild type pneumolysin sequence.
In other embodiments the pneumolysin has a modified pneumolysin
sequence. In yet other embodiments the modified pneumolysin
sequence includes a D205R mutation.
[0012] The Streptococcal vaccine in some embodiments has at least
one RNA polynucleotide with a nucleic acid sequence that has at
least 80%, 85%, 90%, 95%, 98%, or 99% identity to any one of SEQ ID
NO: 6-8, but does not include wild-type mRNA sequence. In other
embodiments the Streptococcal antigenic polypeptide has an amino
acid sequence that has at least 90%, 95%, 98%, or 99% identity to
an amino acid sequence identified by any one of SEQ ID NO: 10-29,
but does not include wild-type protein sequence. In yet other
embodiments the Streptococcal antigenic polypeptide has an amino
acid sequence of any one of SEQ ID NO: 10-29. In some embodiments
the at least one RNA polynucleotide has a nucleic acid sequence of
any one of SEQ ID NO: 6-8.
[0013] Provided herein, in some embodiments, is a ribonucleic acid
(RNA) (e.g., mRNA) vaccine, comprising at least one (e.g., at least
2, 3, 4 or 5) RNA (e.g., mRNA) polynucleotide having an open
reading frame encoding at least one bacterial antigenic
polypeptide, or any combination of two or more of the foregoing
antigenic polypeptides. Herein, use of the term "antigenic
polypeptide" encompasses immunogenic fragments of the antigenic
polypeptide (an immunogenic fragment that is induces (or is capable
of inducing) an immune response to a bacterial infection, unless
otherwise stated.
[0014] Also provided herein, in some embodiments, is a RNA (e.g.,
mRNA) vaccine comprising at least one (e.g., at least 2, 3, 4 or 5)
RNA polynucleotide having an open reading frame encoding at least
one (e.g., at least 2, 3, 4 or 5) bacterial antigenic polypeptide
or an immunogenic fragment thereof, optionally linked to a signal
peptide.
[0015] Further still, provided herein, in some embodiments, is a
method of inducing an immune response in a subject, the method
comprising administering to the subject a vaccine comprising at
least one (e.g., at least 2, 3, 4 or 5) RNA (e.g., mRNA)
polynucleotide having an open reading frame encoding at least one
(e.g., at least 2, 3, 4 or 5) bacterial antigenic polypeptide, or
any combination of two or more of the foregoing antigenic
polypeptides.
[0016] In some embodiments, at least one antigenic polypeptide is a
bacterial polyprotein. In some embodiments, at least one antigenic
polypeptide is pneumolysin or a pneumolysin variant
(pneumolysoid).
[0017] In some embodiments, at least one bacterial antigenic
polypeptide comprises an amino acid sequence identified by any one
of SEQ ID NO: 10-29, 36-38 (Table 2). In some embodiments, the
amino acid sequence of the bacterial antigenic polypeptide is, or
is a fragment of, or is a homolog or variant having at least 80%
(e.g., 85%, 90%, 95%, 98%, 99%, 80-90%, 90-95%, 90-98%, 90-99%,
80-99%) identity to, the amino acid sequence identified by any one
of SEQ ID NO: 10-29, 36-38 (Table 2).
[0018] In some embodiments, the at least one bacterial antigenic
polypeptide is encoded by a nucleic acid sequence from Table 1. In
some embodiments, at least one bacterial antigenic polypeptide is
encoded by a nucleic acid sequence identified by any one of SEQ ID
NO: 2-4, 30-32, 56-61 (Table 1).
[0019] In some embodiments, at least one bacterial RNA (e.g., mRNA)
polynucleotide is encoded by a nucleic acid sequence, or a fragment
of a nucleotide sequence, identified by any one of SEQ ID NO: 2-4,
30-32, 56-61 (Table 1).
[0020] In some embodiments, an open reading frame of a RNA (e.g.,
mRNA) vaccine is codon-optimized. In some embodiments, at least one
RNA polynucleotide encodes at least one antigenic polypeptide
having an amino acid sequence identified by any one of SEQ ID NO:
10-29 (Table 2) and is codon optimized mRNA.
[0021] In some embodiments, a RNA (e.g., mRNA) vaccine further
comprises an adjuvant.
[0022] Each of the amino acid sequences, and variants having
greater than 95% identity or greater than 98% identity to each of
the amino acid sequences encompassed by the accession numbers of
SEQ ID NO: 9 are included within the constructs
(polynucleotides/polypeptides) of the present disclosure.
[0023] In some embodiments, at least one mRNA polynucleotide is
encoded by a nucleic acid having a sequence identified by any one
of SEQ ID NO: 6-8, 33-35, 62-67 (Table 1) and having less than 80%
identity to wild-type mRNA sequence. In some embodiments, at least
one mRNA polynucleotide is encoded by a nucleic acid having a
sequence identified by any one of SEQ ID NO: 6-8, 33-35, 62-67
(Table 1) and having less than 100% but greater than 75%, 85% or
95% identity to a wild-type mRNA sequence. In some embodiments, at
least one mRNA polynucleotide is encoded by a nucleic acid having a
sequence identified by any one of SEQ ID NO: 6-8, 33-35, 62-67
(Table 1) and having 50-80%, 60-80%, 40-80%, 30-80%, 70-80%, 75-80%
or 78-80% identity to wild-type mRNA sequence. In some embodiments,
at least one mRNA polynucleotide is encoded by a nucleic acid
having a sequence identified by any one of SEQ ID NO: 6-8, 33-35,
62-67 (Table 1) and having 40-85%, 50-85%, 60-85%, 30-85%, 70-85%,
75-85% or 80-85% identity to wild-type mRNA sequence. In some
embodiments, at least one mRNA polynucleotide is encoded by a
nucleic acid having a sequence identified by any one of SEQ ID NO:
6-8, 33-35, 62-67 (Table 1) and having 40-90%, 50-90%, 60-90%,
30-90%, 70-90%, 75-90%, 80-90%, or 85-90% identity to wild-type
mRNA sequence.
[0024] In some embodiments, at least one RNA polynucleotide encodes
at least one antigenic polypeptide having an amino acid sequence
identified by any one of SEQ ID NO: 10-29 (Table 2) and having at
least 80% (e.g., 85%, 90%, 95%, 98%, 99%) identity to wild-type
mRNA sequence, but does not include wild-type mRNA sequence.
[0025] In some embodiments, at least one RNA polynucleotide encodes
at least one antigenic polypeptide having an amino acid sequence
identified by any one of SEQ ID NO: 10-29 (Table 2) and has greater
than 95%, 90%, 85%, 80% or 75% identity to wild-type mRNA sequence.
In some embodiments, at least one RNA polynucleotide encodes at
least one antigenic polypeptide having an amino acid sequence
identified by any one of SEQ ID NO: 10-29 (Table 2) and has 30-80%,
40-80%, 50-80%, 60-80%, 70-80%, 75-80% or 78-80%, 30-85%, 40-85%,
50-85%, 60-85%, 70-85%, 75-85% or 78-85%, 30-90%, 40-90%, 50-90%,
60-90%, 70-90%, 75-90%, 80-90% or 85-90% identity to wild-type mRNA
sequence.
[0026] In some embodiments, at least one RNA polynucleotide encodes
at least one antigenic polypeptide having at least 90%, at least
95%, at least 96%, at least 97%, at least 98%, or at least 99%
identity to an amino acid sequence identified by any one of SEQ ID
NO: 10-29 (Table 2). In some embodiments, at least one RNA
polynucleotide encodes at least one antigenic polypeptide having
95%-99% identity to an amino acid sequence identified by any one of
SEQ ID NO: 10-29 (Table 2).
[0027] Some embodiments of the present disclosure provide a vaccine
that includes at least one ribonucleic acid (RNA) (e.g., mRNA)
polynucleotide having an open reading frame encoding at least one
antigenic polypeptide (e.g., at least one bacterial antigenic
polypeptide), at least one 5' terminal cap and at least one
chemical modification, formulated within a lipid nanoparticle.
[0028] In some embodiments, a 5' terminal cap is
7mG(5')ppp(5')NlmpNp.
[0029] In some embodiments, at least one chemical modification is
selected from pseudouridine, N1-methylpseudouridine, 2-thiouridine,
4'-thiouridine, 5-methylcytosine, 5-methyluridine,
2-thio-1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,
2-thio-dihydropseudouridine, 2-thio-dihydrouridine,
2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,
4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,
4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,
5-methoxyuridine and 2'-O-methyl uridine. In some embodiments, the
chemical modification is in the 5-position of the uracil. In some
embodiments, the chemical modification is a
N1-methylpseudouridine.
[0030] In some embodiments, a lipid nanoparticle comprises a
cationic lipid, a PEG-modified lipid, a sterol and a non-cationic
lipid. In some embodiments, a cationic lipid is an ionizable
cationic lipid and the non-cationic lipid is a neutral lipid, and
the sterol is a cholesterol. In some embodiments, a cationic lipid
is selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).
[0031] In some embodiments, a lipid nanoparticle comprises
compounds of Formula (I) and/or Formula (II), discussed below.
[0032] In some embodiments, a bacterial RNA (e.g., mRNA) vaccine is
formulated in a lipid nanoparticle that comprises a compound
selected from Compounds 3, 18, 20, 25, 26, 29, 30, 60, 108-112 and
122, described below.
[0033] Some embodiments of the present disclosure provide a vaccine
that includes at least one RNA (e.g., mRNA) polynucleotide having
an open reading frame encoding at least one antigenic polypeptide
(e.g., at least one bacterial antigenic polypeptide), wherein at
least 80% (e.g., 85%, 90%, 95%, 98%, 99%) of the uracil in the open
reading frame have a chemical modification, optionally wherein the
vaccine is formulated in a lipid nanoparticle (e.g., a lipid
nanoparticle comprises a cationic lipid, a PEG-modified lipid, a
sterol and a non-cationic lipid).
[0034] In some embodiments, 100% of the uracil in the open reading
frame have a chemical modification. In some embodiments, a chemical
modification is in the 5-position of the uracil. In some
embodiments, a chemical modification is a N1-methyl pseudouridine.
In some embodiments, 100% of the uracil in the open reading frame
have a N1-methyl pseudouridine in the 5-position of the uracil.
[0035] In some embodiments, an open reading frame of a RNA (e.g.,
mRNA) polynucleotide encodes at least two antigenic polypeptides
(e.g., at least two bacterial antigenic polypeptides). In some
embodiments, the at least two bacterial antigenic polypeptides are
the same bacterial antigenic polypeptides. In other embodiments,
the at least two bacterial antigenic polypeptides are different
bacterial antigenic polypeptides. In some embodiments, the open
reading frame encodes at least five or at least ten antigenic
polypeptides. In some embodiments, the open reading frame encodes
at least 100 antigenic polypeptides. In some embodiments, the open
reading frame encodes 2-100 antigenic polypeptides.
[0036] In some embodiments, a vaccine comprises at least two RNA
(e.g., mRNA) polynucleotides, each having an open reading frame
encoding at least one antigenic polypeptide (e.g., at least one
bacterial antigenic polypeptide). In some embodiments, the vaccine
comprises at least five or at least ten RNA (e.g., mRNA)
polynucleotides, each having an open reading frame encoding at
least one antigenic polypeptide or an immunogenic fragment thereof.
In some embodiments, the vaccine comprises at least 100 RNA (e.g.,
mRNA) polynucleotides, each having an open reading frame encoding
at least one antigenic polypeptide. In some embodiments, the
vaccine comprises 2-100 RNA (e.g., mRNA) polynucleotides, each
having an open reading frame encoding at least one antigenic
polypeptide.
[0037] In some embodiments, at least one antigenic polypeptide
(e.g., at least one bacterial antigenic polypeptide) is fused to a
signal peptide. In some embodiments, the signal peptide is selected
from: a HulgGk signal peptide (METPAQLLFLLLLWLPDTTG; SEQ ID NO:
39); IgE heavy chain epsilon-1 signal peptide (MDWTWILFLVAAATRVHS;
SEQ ID NO: 40); Japanese encephalitis PRM signal sequence
(MLGSNSGQRVVFTILLLLVAPAYS; SEQ ID NO: 41), VSVg protein signal
sequence (MKCLLYLAFLFIGVNCA; SEQ ID NO: 42) and Japanese
encephalitis JEV signal sequence (MWLVSLAIVTACAGA; SEQ ID NO:
43).
[0038] In some embodiments, the signal peptide is fused to the
N-terminus of at least one antigenic polypeptide. In some
embodiments, a signal peptide is fused to the C-terminus of at
least one antigenic polypeptide.
[0039] Also provided herein is a RNA (e.g., mRNA) vaccine of any
embodiments of the invention formulated in a nanoparticle (e.g., a
lipid nanoparticle or cationic lipid nanoparticle).
[0040] In some embodiments, the nanoparticle has a mean diameter of
50-200 nm. In some embodiments, the nanoparticle is a lipid
nanoparticle. In some embodiments, the lipid nanoparticle comprises
a cationic lipid, a PEG-modified lipid, a sterol and a non-cationic
lipid. In some embodiments, the lipid nanoparticle comprises a
molar ratio of about 20-60% cationic lipid, 0.5-15% PEG-modified
lipid, 25-55% sterol, and 5-25% non-cationic lipid. In some
embodiments, the cationic lipid is an ionizable cationic lipid and
the non-cationic lipid is a neutral lipid, and the sterol is a
cholesterol. In some embodiments, the cationic lipid is selected
from 2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane
(DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate
(DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).
[0041] In some embodiments, a lipid nanoparticle comprises
compounds of Formula (I) and/or Formula (II), as discussed
below.
[0042] In some embodiments, a lipid nanoparticle comprises
Compounds 3, 18, 20, 25, 26, 29, 30, 60, 108-112, or 122, as
discussed below.
[0043] In some embodiments, the nanoparticle has a polydispersity
value of less than 0.4 (e.g., less than 0.3, 0.2 or 0.1).
[0044] In some embodiments, the nanoparticle has a net neutral
charge at a neutral pH value.
[0045] In some embodiments, the bacterial vaccine is
multivalent.
[0046] Some embodiments of the present disclosure provide methods
of inducing an antigen specific immune response in a subject,
comprising administering to the subject any of the RNA (e.g., mRNA)
vaccine as provided herein in an amount effective to produce an
antigen-specific immune response. In some embodiments, the RNA
(e.g., mRNA) vaccine is a bacterial vaccine. In other embodiments,
the RNA (e.g., mRNA) vaccine is a streptococcal vaccine.
[0047] In some embodiments, an antigen-specific immune response
comprises a T cell response or a B cell response.
[0048] In some embodiments, a method of producing an
antigen-specific immune response comprises administering to a
subject a single dose (no booster dose) of a RNA (e.g., mRNA)
vaccine of the present disclosure. In some embodiments, a method
further comprises administering to the subject a second (booster)
dose of a RNA (e.g., mRNA) vaccine. Additional doses of a RNA
(e.g., mRNA) vaccine may be administered.
[0049] In some embodiments, the subjects exhibit a seroconversion
rate of at least 80% (e.g., at least 85%, at least 90%, or at least
95%) following the first dose or the second (booster) dose of the
vaccine. Seroconversion is the time period during which a specific
antibody develops and becomes detectable in the blood. After
seroconversion has occurred, a bacteria can be detected in blood
tests for the antibody. During an infection or immunization,
antigens enter the blood, and the immune system begins to produce
antibodies in response. Before seroconversion, the antigen itself
may or may not be detectable, but antibodies are considered absent.
During seroconversion, antibodies are present but not yet
detectable. Any time after seroconversion, the antibodies can be
detected in the blood, indicating a prior or current infection.
[0050] In some embodiments, an RNA (e.g., mRNA) vaccine is
administered to a subject by intradermal or intramuscular
injection.
[0051] Some embodiments of the present disclosure provide methods
of inducing an antigen specific immune response in a subject,
including administering to a subject a RNA (e.g., mRNA) vaccine in
an effective amount to produce an antigen specific immune response
in a subject. Antigen-specific immune responses in a subject may be
determined, in some embodiments, by assaying for antibody titer
(for titer of an antibody that binds to a bacterial antigenic
polypeptide) following administration to the subject of any of the
RNA (e.g., mRNA) vaccines of the present disclosure. In some
embodiments, the anti-antigenic polypeptide antibody titer produced
in the subject is increased by at least 1 log relative to a
control. In some embodiments, the anti-antigenic polypeptide
antibody titer produced in the subject is increased by 1-3 log
relative to a control.
[0052] In some embodiments, the anti-antigenic polypeptide antibody
titer produced in a subject is increased at least 2 times relative
to a control. In some embodiments, the anti-antigenic polypeptide
antibody titer produced in the subject is increased at least 5
times relative to a control. In some embodiments, the
anti-antigenic polypeptide antibody titer produced in the subject
is increased at least 10 times relative to a control. In some
embodiments, the anti-antigenic polypeptide antibody titer produced
in the subject is increased 2-10 times relative to a control.
[0053] In some embodiments, the control is an anti-antigenic
polypeptide antibody titer produced in a subject who has not been
administered a RNA (e.g., mRNA) vaccine of the present disclosure.
In some embodiments, the control is an anti-antigenic polypeptide
antibody titer produced in a subject who has been administered a
live attenuated or inactivated bacterial vaccine, or wherein the
control is an anti-antigenic polypeptide antibody titer produced in
a subject who has been administered a recombinant or purified
bacterial protein vaccine.
[0054] A RNA (e.g., mRNA) vaccine of the present disclosure is
administered to a subject in an effective amount (an amount
effective to induce an immune response). In some embodiments, the
effective amount is a dose equivalent to an at least 2-fold, at
least 4-fold, at least 10-fold, at least 100-fold, at least
1000-fold reduction in the standard of care dose of a recombinant
bacterial protein vaccine, wherein the anti-antigenic polypeptide
antibody titer produced in the subject is equivalent to an
anti-antigenic polypeptide antibody titer produced in a control
subject administered the standard of care dose of a recombinant
bacterial protein vaccine, a purified bacterial protein vaccine, a
live attenuated bacterial vaccine, or an inactivated bacterial
vaccine. In some embodiments, the effective amount is a dose
equivalent to 2-1000-fold reduction in the standard of care dose of
a recombinant bacterial protein vaccine, wherein the anti-antigenic
polypeptide antibody titer produced in the subject is equivalent to
an anti-antigenic polypeptide antibody titer produced in a control
subject administered the standard of care dose of a recombinant
bacterial protein vaccine, a purified bacterial protein vaccine, a
live attenuated bacterial vaccine, or an inactivated bacterial
vaccine.
[0055] In some embodiments, the RNA (e.g., mRNA) vaccine is
formulated in an effective amount to produce an antigen specific
immune response in a subject.
[0056] In some embodiments, the effective amount is a total dose of
25 .mu.g to 1000 .mu.g, or 50 .mu.g to 1000 .mu.g. In some
embodiments, the effective amount is a total dose of 100 .mu.g. In
some embodiments, the effective amount is a total dose of 1-100
.mu.g. In some embodiments, the effective amount is a dose of 25
.mu.g administered to the subject a total of one or two times. In
some embodiments, the effective amount is a dose of 100 .mu.g
administered to the subject a total of two times. In some
embodiments, the effective amount is a dose of 1 .mu.g-10 .mu.g, 1
.mu.g-20 .mu.g, 1 .mu.g-30 .mu.g, 5 .mu.g-10 .mu.g, 5 .mu.g-20
.mu.g, 5 .mu.g-30 .mu.g, 5 .mu.g-40 .mu.g, 5 .mu.g-50 .mu.g, 10
.mu.g-15 .mu.g, 10 .mu.g-20 .mu.g, 10 .mu.g-25 .mu.g, 10 .mu.g-30
.mu.g, 10 .mu.g-40 .mu.g, 10 .mu.g-50 .mu.g, 10 .mu.g-60 .mu.g, 15
.mu.g-20 .mu.g, 15 .mu.g-25 .mu.g, 15 .mu.g-30 .mu.g, 15 .mu.g-40
.mu.g, 15 .mu.g-50 .mu.g, 20 .mu.g-25 .mu.g, 20 .mu.g-30 .mu.g, 20
.mu.g-40 .mu.g 20 .mu.g-50 .mu.g, 20 .mu.g-60 .mu.g, 20 .mu.g-70
.mu.g, 20 .mu.g-75 .mu.g, 30 .mu.g-35 .mu.g, 30 .mu.g-40 .mu.g, 30
.mu.g-45 .mu.g 30 .mu.g-50 .mu.g, 30 .mu.g-60 .mu.g, 30 .mu.g-70
.mu.g, 30 .mu.g-75 .mu.g, 5 .mu.g-100 .mu.g, 10 .mu.g-100 .mu.g, 15
.mu.g-100 .mu.g, 20 .mu.g-100 .mu.g, 25 .mu.g-100 .mu.g, 25
.mu.g-500 .mu.g, 50 .mu.g-100 .mu.g, 50 .mu.g-500 .mu.g, 50
.mu.g-1000 .mu.g, 100 .mu.g-500 .mu.g, 100 .mu.g-1000 .mu.g, 250
.mu.g-500 .mu.g, 250 .mu.g-1000 .mu.g, or 500 .mu.g-1000 .mu.g
which may be administered to the subject a total of one or two
times or more.
[0057] In some embodiments, the efficacy (or effectiveness) of a
RNA (e.g., mRNA) vaccine is greater than 60%. In some embodiments,
the RNA (e.g., mRNA) polynucleotide of the vaccine comprises at
least one bacterial antigenic polypeptide.
[0058] Vaccine efficacy may be assessed using standard analyses
(see, e.g., Weinberg et al., J Infect Dis. 2010 Jun. 1;
201(11):1607-10). For example, vaccine efficacy may be measured by
double-blind, randomized, clinical controlled trials. Vaccine
efficacy may be expressed as a proportionate reduction in disease
attack rate (AR) between the unvaccinated (ARU) and vaccinated
(ARV) study cohorts and can be calculated from the relative risk
(RR) of disease among the vaccinated group with use of the
following formulas:
Efficacy=(ARU-ARV)/ARU.times.100; and
Efficacy=(1-RR).times.100.
[0059] Likewise, vaccine effectiveness may be assessed using
standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010
Jun. 1; 201(11):1607-10). Vaccine effectiveness is an assessment of
how a vaccine (which may have already proven to have high vaccine
efficacy) reduces disease in a population. This measure can assess
the net balance of benefits and adverse effects of a vaccination
program, not just the vaccine itself, under natural field
conditions rather than in a controlled clinical trial. Vaccine
effectiveness is proportional to vaccine efficacy (potency) but is
also affected by how well target groups in the population are
immunized, as well as by other non-vaccine-related factors that
influence the `real-world` outcomes of hospitalizations, ambulatory
visits, or costs. For example, a retrospective case control
analysis may be used, in which the rates of vaccination among a set
of infected cases and appropriate controls are compared. Vaccine
effectiveness may be expressed as a rate difference, with use of
the odds ratio (OR) for developing infection despite
vaccination:
Effectiveness=(1-OR).times.100.
[0060] In some embodiments, the efficacy (or effectiveness) of a
RNA (e.g., mRNA) vaccine is at least 65%, at least 70%, at least
75%, at least 80%, at least 85%, or at least 90%.
[0061] In some embodiments, the vaccine immunizes the subject
against the bacteria for up to 2 years. In some embodiments, the
vaccine immunizes the subject against the bacteria for more than 2
years, more than 3 years, more than 4 years, or for 5-10 years.
[0062] In some embodiments, the subject is about 5 years old or
younger. For example, the subject may be between the ages of about
1 year and about 5 years (e.g., about 1, 2, 3, 5 or 5 years), or
between the ages of about 6 months and about 1 year (e.g., about 6,
7, 8, 9, 10, 11 or 12 months). In some embodiments, the subject is
about 12 months or younger (e.g., 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,
2 months or 1 month). In some embodiments, the subject is about 6
months or younger.
[0063] In some embodiments, the subject was born full term (e.g.,
about 37-42 weeks). In some embodiments, the subject was born
prematurely, for example, at about 36 weeks of gestation or earlier
(e.g., about 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26 or 25
weeks). For example, the subject may have been born at about 32
weeks of gestation or earlier. In some embodiments, the subject was
born prematurely between about 32 weeks and about 36 weeks of
gestation. In such subjects, a RNA (e.g., mRNA) vaccine may be
administered later in life, for example, at the age of about 6
months to about 5 years, or older.
[0064] In some embodiments, the subject is pregnant (e.g., in the
first, second or third trimester) when administered an RNA (e.g.,
mRNA) vaccine. Bacterial infections may cause infections of the
respiratory tract, mainly in infants and young children. Pneumonia
is the single largest infectious disease cause of death in children
worldwide, killing 920,163 children under the age of 5 in 2015 and
accounting for approximately 16% of the deaths of children under
the age of 5. Thus, the present disclosure provides RNA (e.g.,
mRNA) vaccines for maternal immunization to improve mother-to-child
transmission of protection against the bacteria.
[0065] In some embodiments, the subject is a young adult between
the ages of about 20 years and about 50 years (e.g., about 20, 25,
30, 35, 40, 45 or 50 years old).
[0066] In some embodiments, the subject is an elderly subject about
60 years old, about 70 years old, or older (e.g., about 60, 65, 70,
75, 80, 85 or 90 years old).
[0067] In some embodiments, the subject is has a chronic pulmonary
disease (e.g., chronic obstructive pulmonary disease (COPD) or
asthma). Two forms of COPD include chronic bronchitis, which
involves a long-term cough with mucus, and emphysema, which
involves damage to the lungs over time. Thus, a subject
administered a RNA (e.g., mRNA) vaccine may have chronic bronchitis
or emphysema.
[0068] In some embodiments, the subject has been exposed to one or
more bacteria.
[0069] In some embodiments, the subject is immunocompromised (has
an impaired immune system, e.g., has an immune disorder or
autoimmune disorder).
[0070] In some embodiments the nucleic acid vaccines described
herein are chemically modified. In other embodiments the nucleic
acid vaccines are unmodified.
[0071] Yet other aspects provide compositions for and methods of
vaccinating a subject comprising administering to the subject a
nucleic acid vaccine comprising one or more RNA polynucleotides
having an open reading frame encoding a first bacterial antigenic
polypeptide, wherein the RNA polynucleotide does not include a
stabilization element, and wherein an adjuvant is not coformulated
or co-administered with the vaccine.
[0072] In some embodiments, the RNA polynucleotide accumulates at a
100 fold higher level in the local lymph node in comparison with
the distal lymph node. In other embodiments the nucleic acid
vaccine is chemically modified and in other embodiments the nucleic
acid vaccine is not chemically modified.
[0073] Aspects of the invention provide a nucleic acid vaccine
comprising one or more RNA polynucleotides having an open reading
frame encoding a first antigenic polypeptide, wherein the RNA
polynucleotide does not include a stabilization element, and a
pharmaceutically acceptable carrier or excipient, wherein an
adjuvant is not included in the vaccine. In some embodiments, the
stabilization element is a histone stem-loop. In some embodiments,
the stabilization element is a nucleic acid sequence having
decreased GC content relative to wild type sequence.
[0074] Aspects of the invention provide nucleic acid vaccines
comprising one or more RNA polynucleotides having an open reading
frame encoding a first antigenic polypeptide, wherein the RNA
polynucleotide is present in the formulation for in vivo
administration to a host, which confers an antibody titer superior
to the criterion for seroprotection for the first antigen for an
acceptable percentage of human subjects. In some embodiments, the
antibody titer is a neutralizing antibody titer.
[0075] Also provided are nucleic acid vaccines comprising one or
more RNA polynucleotides having an open reading frame encoding a
first antigenic polypeptide, wherein the RNA polynucleotide is
present in a formulation for in vivo administration to a host for
eliciting a longer lasting high antibody titer than an antibody
titer elicited by an mRNA vaccine having a stabilizing element or
formulated with an adjuvant and encoding the first antigenic
polypeptide. In some embodiments, the RNA polynucleotide is
formulated to produce neutralizing antibodies within one week of a
single administration. In some embodiments, the adjuvant is
selected from a cationic peptide and an immunostimulatory nucleic
acid. In some embodiments, the cationic peptide is protamine.
[0076] Aspects provide nucleic acid vaccines comprising one or more
RNA polynucleotides having an open reading frame comprising at
least one chemical modification or optionally no chemical
modification, the open reading frame encoding a first antigenic
polypeptide, wherein the RNA polynucleotide is present in the
formulation for in vivo administration to a host such that the
level of antigen expression in the host significantly exceeds a
level of antigen expression produced by an mRNA vaccine having a
stabilizing element or formulated with an adjuvant and encoding the
first antigenic polypeptide.
[0077] Other aspects provide nucleic acid vaccines comprising one
or more RNA polynucleotides having an open reading frame comprising
at least one chemical modification or optionally no chemical
modification, the open reading frame encoding a first antigenic
polypeptide, wherein the vaccine has at least 10 fold less RNA
polynucleotide than is required for an unmodified mRNA vaccine to
produce an equivalent antibody titer. In some embodiments, the RNA
polynucleotide is present in a dosage of 25-100 micrograms.
[0078] Aspects of the invention also provide a unit of use vaccine,
comprising between 10 ug and 400 ug of one or more RNA
polynucleotides having an open reading frame comprising at least
one chemical modification or optionally no chemical modification,
the open reading frame encoding a first antigenic polypeptide, and
a pharmaceutically acceptable carrier or excipient, formulated for
delivery to a human subject. In some embodiments, the vaccine
further comprises a cationic lipid nanoparticle.
[0079] The data presented in the Examples demonstrate significant
enhanced immune responses using the formulations of the invention.
The data demonstrated the effectiveness of mutating N-glycosylation
sites in the RNA vaccines of the invention. Surprisingly, it was
discovered herein that N-glycosylation mutated mRNA vaccines has a
positive effect on bacterial neutralization, even when lower levels
of IgG are observed.
[0080] Each of the limitations of the invention can encompass
various embodiments of the invention. It is, therefore, anticipated
that each of the limitations of the invention involving any one
element or combinations of elements can be included in each aspect
of the invention. This invention is not limited in its application
to the details of construction and the arrangement of components
set forth in the following description or illustrated in the
drawings. The invention is capable of other embodiments and of
being practiced or of being carried out in various ways.
BRIEF DESCRIPTION OF THE DRAWINGS
[0081] The foregoing and other objects, features and advantages
will be apparent from the following description of particular
embodiments of the disclosure, as illustrated in the accompanying
drawings in which like reference characters refer to the same parts
throughout the different views. The drawings are not necessarily to
scale, emphasis instead being placed upon illustrating the
principles of various embodiments of the disclosure.
[0082] FIGS. 1A-1B show the results of serum IgG anti-pneumolysin
assays 21 days after the first immunization (FIG. 1A) and 41 days
after the first immunization (FIG. 1B).
[0083] FIG. 2 shows a hemolytic unit (HU) determination curve. The
pneumolysin concentration for 50% hemolysis was found to be 5
ng/mL.
[0084] FIG. 3 shows the results of a serum neutralization assay
using 4 HU. The serum used was from the 41 day, 10 .mu.g dose
groups.
[0085] FIG. 4 shows shows the expression of mRNAs encoding
previously characterized pneumolysin toxoid variants in HEK293F
cells. The pneumolysin-predicted molecular weight was 53 kDa, and
E. coli produced runs approximately 62 kDa. Detection was with
rabbit anti-His pAb (abcam ab9108) (light gray) and mouse anti-beta
actin mAb (dark gray). The lack of expression in the L460D NGM
group was likely due to a technical error, as all variants were
well--expressed and the construct immunogenic in vivo. L, lysate,
S, supernatant, cS, concentrated supernatant, WT, wild type, and
NGM, N-linked glycosylation mutant.
[0086] FIGS. 5A-5B show the expression of mRNA novel pneumolysin
toxoid variants in HEK293F cells. Detection was with rabbit
anti-His pAb (abcam ab9108) (green) and mouse anti-beta actin mAb
(red). L, lysate, S, supernatant, cS, concentrated supernatant, WT,
wild type, and NGM, N-linked glycosylation mutant.
[0087] FIG. 6 shows the results of a serum neutralization assay
using 4 HU. The serum used was from the 41 day, 2 .mu.g and 10
.mu.g dose groups.
[0088] FIG. 7 shows the expression of mRNA PspA variants from the
TIGR4 and Rx1 strains in HEK293 cells. Detection was with rabbit
anti-His (A1647) (light gray) and mouse anti-actin mAb (Cy3) (dark
gray).
[0089] FIG. 8 shows the expression of mRNA CbpA variants from the
TIGR4 strain in HEK293 cells. Instead of using untransfected cells
as a control, positive PspA was used. Detection was with rabbit
anti-His (A1647) (light gray) and mouse anti-actin mAb (Cy3) (dark
gray) (left blot) and with rabbit anti-His (A1647) (right
blot).
[0090] FIG. 9 shows the expression of mRNA PhtD, PiaA, and PiuA
variants in HEK293 cells. Detection was with rabbit anti-His
(A1647) (light gray) and mouse anti-actin mAb (Cy3) (dark
gray).
[0091] FIG. 10 shows the expression of mRNA PcsB and SktP in HEK293
cells. Detection was with rabbit anti-His (A1647) (light gray) and
mouse anti-actin mAb (Cy3) (dark gray).
[0092] FIG. 11 shows the expression of mRNA PsaA and PcpA in HEK293
cells. Detection was with rabbit anti-His (A1647) (light gray) and
mouse anti-actin mAb (Cy3) (dark gray).
[0093] FIG. 12 shows the expression of mRNA StkP and PhtE in HEK293
cells. Detection was with rabbit anti-His (A1647) (light gray) and
mouse anti-actin mAb (Cy3) (dark gray).
[0094] FIG. 13 shows the results of a serum IgG anti-PspA assay on
day 42 of an in vivo immunogenicity study testing PspA vaccine
constructs.
[0095] FIG. 14 shows the results of a serum IgG anti-CbpA assay on
days 20 and 42 of an in vivo immunogenicity study testing CbpA
vaccine constructs.
[0096] FIG. 15 shows the results of a serum IgG anti-PiaA assay on
days 21 and 41 of an in vivo immunogenicity study testing PiaA
vaccine constructs.
[0097] FIG. 16 shows the results of a serum IgG anti-PiuA assay on
days 21 and 41 of an in vivo immunogenicity study testing PiuA
vaccine constructs.
[0098] FIG. 17 shows the results of a serum IgG anti-PhtD assay on
days 22 and 36 of an in vivo immunogenicity study testing PhtD
vaccine constructs.
[0099] FIG. 18 shows the results of six cytokine assays used to
determine the cytokine response in splenic samples.
DETAILED DESCRIPTION
[0100] The bacterial RNA vaccines described herein are superior to
current vaccines in several ways. For example, the lipid
nanoparticle (LNP) delivery system used herein increases the
efficacy of RNA vaccines in comparison to other formulations,
including a protamine-based approach described in the literature.
The use of this LNP delivery system enables the effective delivery
of chemically-modified RNA vaccines or unmodified RNA vaccines,
without requiring additional adjuvant to produce a therapeutic
result (e.g., production neutralizing antibody titer and/or a T
cell response). In some embodiments, the bacterial RNA vaccines
disclosed herein are superior to conventional vaccines by a factor
of at least 10 fold, 20, fold, 40, fold, 50 fold, 100 fold, 500
fold, or 1,000 fold when administered intramuscularly (IM) or
intradermally (ID). These results can be achieved even when
significantly lower doses of the RNA (e.g., mRNA) are administered
in comparison with RNA doses used in other classes of lipid based
formulations.
[0101] The LNP used in the studies described herein has been used
previously to deliver siRNA in various animal models as well as in
humans. In view of the observations made in association with the
siRNA delivery of LNP formulations, the fact that LNP is useful in
vaccines is quite surprising, particularly when immunity to an
antigen has been hard to generate, as in the case of bacterial
infections. It has been observed that therapeutic delivery of siRNA
formulated in LNP causes an undesirable inflammatory response
associated with a transient IgM response, typically leading to a
reduction in antigen production and a compromised immune response.
In contrast to the findings observed with siRNA, the LNP-mRNA
formulations of the present disclosure are demonstrated herein to
generate enhanced IgG levels, sufficient for prophylactic and
therapeutic methods rather than transient IgM responses.
[0102] The present disclosure provides, in some embodiments,
vaccines that comprise RNA (e.g., mRNA) polynucleotides encoding a
bacterial antigenic polypeptide. Also provided herein are methods
of administering the RNA (e.g., mRNA) vaccines, methods of
producing the RNA (e.g., mRNA) vaccines, compositions (e.g.,
pharmaceutical compositions) comprising the RNA (e.g., mRNA)
vaccines, and nucleic acids (e.g., DNA) encoding the RNA (e.g.,
mRNA) vaccines. In some embodiments, a RNA (e.g., mRNA) vaccine
comprises an adjuvant, such as a flagellin adjuvant, as provided
herein.
[0103] The RNA (e.g., mRNA) bacterial vaccines, in some
embodiments, may be used to induce a balanced immune response,
comprising both cellular and humoral immunity, without many of the
risks associated with DNA vaccination.
[0104] Although attempts have been made to produce functional
recombinant bacterial vaccines, the therapeutic efficacy of these
vaccines have not yet been fully established. Quite surprisingly,
the inventors have discovered, according to aspects of the
invention that a class of formulations for delivering mRNA vaccines
in vivo that results in significantly enhanced, immune responses
including enhanced neutralization capability. The formulations of
the invention have demonstrated significant unexpected immune
responses sufficient to establish the efficacy of functional
bacterial mRNA vaccines as prophylactic and therapeutic agents.
[0105] In certain aspects, the invention provides a polypeptide
comprising mutated N-linked glycosylation sites. N-linked glycans
of bacterial proteins play important roles in modulating the immune
response. Glycans can be important for maintaining the appropriate
antigenic conformations, shielding potential neutralization
epitopes, and may alter the proteolytic susceptibility of proteins.
Some bacteria have putative N-linked glycosylation sites. Deletion
or modification of an N-linked glycosylation site may enhance the
immune response. Thus, the present disclosure provides, in some
embodiments, RNA (e.g., mRNA) vaccines comprising nucleic acids
(e.g., mRNA) encoding antigenic polypeptides that comprise a
deletion or modification at one or more N-linked glycosylation
sites. N-linked glycosylation, the attachment of a glycan (sugar
molecule oligosaccharide) to the amide nitrogen of an asparagine
residue, occurs frequently in eukaryotes but rarely in bacteria.
Imperiali et al., Curr Opin in Chem Biol. 3(6):643-649 (1999).
Without wishing to be bound by any theory, it is thought that
expressing the bacterial protein in a mammalian cell may lead to
glycosylated antigens having altered or ineffective immunogenicity
profiles. Thus, in some embodiments, the pneumolysin polypeptide
comprises one or more n-linked glycosylation site(s).
[0106] Pneumolysin (PLY, AJS15225.1; M17717.1), a putative major
virulence factor of Streptococcus pneumoniae, is a 53 kDa
pore-forming toxin consisting of 471 amino acids. Marriott et al.,
Curr Mol Med. 8(6):497-509 (2008). The toxin is inhibited by
cholesterol, and at high levels (greater than 50 hemolytic units),
it is lytic to all cells with cholesterol in the membrane. At
lower, sublytic concentrations, pneumolysin can induce apoptosis,
activate the host complement, and induce proinflammatory reactions
in immune cells. Pneumolysin is generally located in the bacterial
cytoplasm, but does not have an N-terminal secretion signal
sequence, so it is released when the pneumococcus undergoes
autolysis with N-acetyl-muramoyl-1-alanine amidase (Lyt A). Hirst
et al., Clin Exp Immunol. 138(2): 195-201 (2004). The toxin, a
water-soluble monomer, recognizes mammalian cells via its
C-terminal domain (domain 4), and assembles into circular prepores
of approximately 30-50 monomers on the surface of cholesterol-rich
membranes. When bound, the monomers undergo conformational changes,
resulting in a PLY .beta.-barrel pore that causes lysis of the
target cell. Lawrence et al., Sci Rep. 5:14352 (2015).
[0107] The invention in aspects is a composition of an RNA
polynucleotide comprising an open reading frame (ORF) encoding
pneumolysin (PLY) polypeptide which may be formulated in a cationic
lipid nanoparticle. The PLY may be a wild type PLY or a variant
polypeptide. The compositions of the invention have several
advantages over prior art methods for treating pneumococcal
infections, including prior art PLY formulations such as protein or
nucleic acid PLY formulations.
[0108] Bacterial vaccines, as provided herein, comprise at least
one (one or more) ribonucleic acid (RNA) (e.g., mRNA)
polynucleotide having an open reading frame encoding at least one
mutated antigenic polypeptide selected from Acetobacter,
Acinetobacter, Actinomyces, Agrobacterium, Anaplasma, Azorhizobia,
Bacillus, Bacteroides, Bartonella, Bordetella, Borrelia, Brucella,
Burkkolderia, Calymmatobacterium, Campylobacter, Chlamydia,
Chlamydophila, Clostridium, Corynebacterium, Coxiella, Ehrlichia,
Enterobacter, Enterococcus, Escherichia, Francisella,
Fusobacterium, Gardnerella, Haemophilus, Helicobacter, Klebsiella,
Lactobacillus, Legionella, Listeria, Methanobacterium,
Microbacterium, Micrococcus, Moraxella, Mycobacterium, Mycoplasma,
Neisseria, Pasteurella, Peptostreptococcus, Porphyromonas,
Prevotella, Pseudomonas, Rhizobium, Rickettsia, Rochalimaea,
Rothia, Salmonella, Shigella, Staphylococcus, Stenotrophomonas,
Streptococcus, Treponema, Vibrio, Walbachia, and Yersinia antigenic
polypeptides and containing at least one N-linked glycosylation
mutation. The term "nucleic acid" includes any compound and/or
substance that comprises a polymer of nucleotides (nucleotide
monomer). These polymers are referred to as polynucleotides. Thus,
the terms "nucleic acid" and "polynucleotide" are used
interchangeably.
[0109] Nucleic acids may be or may include, for example,
ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose
nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic
acids (PNAs), locked nucleic acids (LNAs, including LNA having a
.beta.-D-ribo configuration, .alpha.-LNA having an .alpha.-L-ribo
configuration (a diastereomer of LNA), 2'-amino-LNA having a
2'-amino functionalization, and 2'-amino-.alpha.-LNA having a
2'-amino functionalization), ethylene nucleic acids (ENA),
cyclohexenyl nucleic acids (CeNA) or chimeras or combinations
thereof.
[0110] In some embodiments, polynucleotides of the present
disclosure function as messenger RNA (mRNA). "Messenger RNA" (mRNA)
refers to any polynucleotide that encodes a (at least one)
polypeptide (a naturally-occurring, non-naturally-occurring, or
modified polymer of amino acids) and can be translated to produce
the encoded polypeptide in vitro, in vivo, in situ or ex vivo. The
skilled artisan will appreciate that, except where otherwise noted,
polynucleotide sequences set forth in the instant application will
recite "T"s in a representative DNA sequence but where the sequence
represents RNA (e.g., mRNA), the "T"s would be substituted for
"U"s. Thus, any of the RNA polynucleotides encoded by a DNA
identified by a particular sequence identification number may also
comprise the corresponding RNA (e.g., mRNA) sequence encoded by the
DNA, where each "T" of the DNA sequence is substituted with
"U."
[0111] The basic components of an mRNA molecule typically include
at least one coding region, a 5' untranslated region (UTR), a 3'
UTR, a 5' cap and a poly-A tail. Polynucleotides of the present
disclosure may function as mRNA but can be distinguished from
wild-type mRNA in their functional and/or structural design
features, which serve to overcome existing problems of effective
polypeptide expression using nucleic-acid based therapeutics.
[0112] In some embodiments, a RNA polynucleotide of an RNA (e.g.,
mRNA) vaccine encodes 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3,
3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5,
5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8,
8-10, 8-9 or 9-10 antigenic polypeptides. In some embodiments, a
RNA (e.g., mRNA) polynucleotide of a bacterial vaccine encodes at
least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 antigenic
polypeptides. In some embodiments, a RNA (e.g., mRNA)
polynucleotide of a bacterial vaccine encodes at least 100 or at
least 200 antigenic polypeptides. In some embodiments, a RNA
polynucleotide of bacterial vaccine encodes 1-10, 5-15, 10-20,
15-25, 20-30, 25-35, 30-40, 35-45, 40-50, 1-50, 1-100, 2-50 or
2-100 antigenic polypeptides.
Codon Optimization
[0113] Polynucleotides of the present disclosure, in some
embodiments, are codon optimized. Codon optimization methods are
known in the art and may be used as provided herein. Codon
optimization, in some embodiments, may be used to match codon
frequencies in target and host organisms to ensure proper folding;
bias GC content to increase mRNA stability or reduce secondary
structures; minimize tandem repeat codons or base runs that may
impair gene construction or expression; customize transcriptional
and translational control regions; insert or remove protein
trafficking sequences; remove/add post translation modification
sites in encoded protein (e.g. glycosylation sites); add, remove or
shuffle protein domains; insert or delete restriction sites; modify
ribosome binding sites and mRNA degradation sites; adjust
translational rates to allow the various domains of the protein to
fold properly; or to reduce or eliminate problem secondary
structures within the polynucleotide. Codon optimization tools,
algorithms and services are known in the art--non-limiting examples
include services from GeneArt (Life Technologies), DNA2.0 (Menlo
Park Calif.) and/or proprietary methods. In some embodiments, the
open reading frame (ORF) sequence is optimized using optimization
algorithms.
[0114] In some embodiments, a codon optimized sequence shares less
than 95% sequence identity, less than 90% sequence identity, less
than 85% sequence identity, less than 80% sequence identity, or
less than 75% sequence identity to a naturally-occurring or
wild-type sequence (e.g., a naturally-occurring or wild-type mRNA
sequence encoding a polypeptide or protein of interest (e.g., an
antigenic protein or antigenic polypeptide)).
[0115] In some embodiments, a codon-optimized sequence shares
between 65% and 85% (e.g., between about 67% and about 85%, or
between about 67% and about 80%) sequence identity to a
naturally-occurring sequence or a wild-type sequence (e.g., a
naturally-occurring or wild-type mRNA sequence encoding a
polypeptide or protein of interest (e.g., an antigenic protein or
polypeptide)). In some embodiments, a codon-optimized sequence
shares between 65% and 75%, or about 80% sequence identity to a
naturally-occurring sequence or wild-type sequence (e.g., a
naturally-occurring or wild-type mRNA sequence encoding a
polypeptide or protein of interest (e.g., an antigenic protein or
polypeptide)).
[0116] In some embodiments, a codon-optimized sequence encodes an
antigen that is as immunogenic as, or more immunogenic than (e.g.,
at least 10%, at least 20%, at least 30%, at least 40%, at least
50%, at least 100%, or at least 200% more), than an XXX antigen
encoded by a non-codon-optimized sequence.
[0117] When transfected into mammalian host cells, the modified
mRNAs have a stability of between 12-18 hours, or greater than 18
hours, e.g., 24, 36, 48, 60, 72, or greater than 72 hours and are
capable of being expressed by the mammalian host cells.
[0118] In some embodiments a codon-optimized RNA (e.g., mRNA) may,
for instance, be one in which the levels of G/C are enhanced. The
G/C-content of nucleic acid molecules may influence the stability
of the RNA. RNA having an increased amount of guanine (G) and/or
cytosine (C) residues may be functionally more stable than nucleic
acids containing a large amount of adenine (A) and thymine (T) or
uracil (U) nucleotides. WO02/098443 discloses a pharmaceutical
composition containing an mRNA stabilized by sequence modifications
in the translated region. Due to the degeneracy of the genetic
code, the modifications work by substituting existing codons for
those that promote greater RNA stability without changing the
resulting amino acid. The approach is limited to coding regions of
the RNA.
[0119] In some embodiments, an antigenic polypeptide is longer than
25 amino acids and shorter than 50 amino acids. Polypeptides
include gene products, naturally occurring polypeptides, synthetic
polypeptides, homologs, orthologs, paralogs, fragments and other
equivalents, variants, and analogs of the foregoing. A polypeptide
may be a single molecule or may be a multi-molecular complex such
as a dimer, trimer or tetramer. Polypeptides may also comprise
single chain polypeptides or multichain polypeptides, such as
antibodies or insulin, and may be associated or linked to each
other. Most commonly, disulfide linkages are found in multichain
polypeptides. The term "polypeptide" may also apply to amino acid
polymers in which at least one amino acid residue is an artificial
chemical analogue of a corresponding naturally-occurring amino
acid.
[0120] A "polypeptide variant" is a molecule that differs in its
amino acid sequence relative to a native sequence or a reference
sequence. Amino acid sequence variants may possess substitutions,
deletions, insertions, or a combination of any two or three of the
foregoing, at certain positions within the amino acid sequence, as
compared to a native sequence or a reference sequence. Ordinarily,
variants possess at least 50% identity to a native sequence or a
reference sequence. In some embodiments, variants share at least
80% identity or at least 90% identity with a native sequence or a
reference sequence.
[0121] Variant antigens/polypeptides encoded by nucleic acids of
the disclosure may contain amino acid changes that confer any of a
number of desirable properties, e.g., that enhance their
immunogenicity, enhance their expression, and/or improve their
stability or PK/PD properties in a subject. Variant
antigens/polypeptides can be made using routine mutagenesis
techniques and assayed as appropriate to determine whether they
possess the desired property. Assays to determine expression levels
and immunogenicity are well known in the art and exemplary such
assays are set forth in the Examples section. Similarly, PK/PD
properties of a protein variant can be measured using art
recognized techniques, e.g., by determining expression of antigens
in a vaccinated subject over time and/or by looking at the
durability of the induced immune response. The stability of
protein(s) encoded by a variant nucleic acid may be measured by
assaying thermal stability or stability upon urea denaturation or
may be measured using in silico prediction. Methods for such
experiments and in silico determinations are known in the art.
[0122] In some embodiments "variant mimics" are provided. A
"variant mimic" contains at least one amino acid that would mimic
an activated sequence. For example, glutamate may serve as a mimic
for phosphoro-threonine and/or phosphoro-serine. Alternatively,
variant mimics may result in deactivation or in an inactivated
product containing the mimic. For example, phenylalanine may act as
an inactivating substitution for tyrosine, or alanine may act as an
inactivating substitution for serine.
[0123] "Orthologs" refers to genes in different species that
evolved from a common ancestral gene by speciation. Normally,
orthologs retain the same function in the course of evolution.
Identification of orthologs is important for reliable prediction of
gene function in newly sequenced genomes.
[0124] "Analogs" is meant to include polypeptide variants that
differ by one or more amino acid alterations, for example,
substitutions, additions or deletions of amino acid residues that
still maintain one or more of the properties of the parent or
starting polypeptide.
[0125] The present disclosure provides several types of
compositions that are polynucleotide or polypeptide based,
including variants and derivatives. These include, for example,
substitutional, insertional, deletion and covalent variants and
derivatives. The term "derivative" is synonymous with the term
"variant" and generally refers to a molecule that has been modified
and/or changed in any way relative to a reference molecule or a
starting molecule.
[0126] As such, polynucleotides encoding peptides or polypeptides
containing substitutions, insertions and/or additions, deletions
and covalent modifications with respect to reference sequences, in
particular the polypeptide sequences disclosed herein, are included
within the scope of this disclosure. For example, sequence tags or
amino acids, such as one or more lysines, can be added to peptide
sequences (e.g., at the N-terminal or C-terminal ends). Sequence
tags can be used for peptide detection, purification or
localization. Lysines can be used to increase peptide solubility or
to allow for biotinylation. Alternatively, amino acid residues
located at the carboxy and amino terminal regions of the amino acid
sequence of a peptide or protein may optionally be deleted
providing for truncated sequences. Certain amino acids (e.g.,
C-terminal residues or N-terminal residues) alternatively may be
deleted depending on the use of the sequence, as for example,
expression of the sequence as part of a larger sequence that is
soluble, or linked to a solid support.
[0127] "Substitutional variants" when referring to polypeptides are
those that have at least one amino acid residue in a native or
starting sequence removed and a different amino acid inserted in
its place at the same position. Substitutions may be single, where
only one amino acid in the molecule has been substituted, or they
may be multiple, where two or more (e.g., 3, 4 or 5) amino acids
have been substituted in the same molecule.
[0128] As used herein the term "conservative amino acid
substitution" refers to the substitution of an amino acid that is
normally present in the sequence with a different amino acid of
similar size, charge, or polarity. Examples of conservative
substitutions include the substitution of a non-polar (hydrophobic)
residue such as isoleucine, valine and leucine for another
non-polar residue. Likewise, examples of conservative substitutions
include the substitution of one polar (hydrophilic) residue for
another such as between arginine and lysine, between glutamine and
asparagine, and between glycine and serine. Additionally, the
substitution of a basic residue such as lysine, arginine or
histidine for another, or the substitution of one acidic residue
such as aspartic acid or glutamic acid for another acidic residue
are additional examples of conservative substitutions. Examples of
non-conservative substitutions include the substitution of a
non-polar (hydrophobic) amino acid residue such as isoleucine,
valine, leucine, alanine, methionine for a polar (hydrophilic)
residue such as cysteine, glutamine, glutamic acid or lysine and/or
a polar residue for a non-polar residue.
[0129] "Features" when referring to polypeptide or polynucleotide
are defined as distinct amino acid sequence-based or
nucleotide-based components of a molecule respectively. Features of
the polypeptides encoded by the polynucleotides include surface
manifestations, local conformational shape, folds, loops,
half-loops, domains, half-domains, sites, termini and any
combination(s) thereof.
[0130] As used herein when referring to polypeptides the term
"domain" refers to a motif of a polypeptide having one or more
identifiable structural or functional characteristics or properties
(e.g., binding capacity, serving as a site for protein-protein
interactions).
[0131] As used herein when referring to polypeptides the terms
"site" as it pertains to amino acid based embodiments is used
synonymously with "amino acid residue" and "amino acid side chain."
As used herein when referring to polynucleotides the terms "site"
as it pertains to nucleotide based embodiments is used synonymously
with "nucleotide." A site represents a position within a peptide or
polypeptide or polynucleotide that may be modified, manipulated,
altered, derivatized or varied within the polypeptide-based or
polynucleotide-based molecules.
[0132] As used herein the terms "termini" or "terminus" when
referring to polypeptides or polynucleotides refers to an extremity
of a polypeptide or polynucleotide respectively. Such extremity is
not limited only to the first or final site of the polypeptide or
polynucleotide but may include additional amino acids or
nucleotides in the terminal regions. Polypeptide-based molecules
may be characterized as having both an N-terminus (terminated by an
amino acid with a free amino group (NH2)) and a C-terminus
(terminated by an amino acid with a free carboxyl group (COOH)).
Proteins are in some cases made up of multiple polypeptide chains
brought together by disulfide bonds or by non-covalent forces
(multimers, oligomers). These proteins have multiple N- and
C-termini. Alternatively, the termini of the polypeptides may be
modified such that they begin or end, as the case may be, with a
non-polypeptide based moiety such as an organic conjugate.
[0133] As recognized by those skilled in the art, protein
fragments, functional protein domains, and homologous proteins are
also considered to be within the scope of polypeptides of interest.
For example, provided herein is any protein fragment (meaning a
polypeptide sequence at least one amino acid residue shorter than a
reference polypeptide sequence but otherwise identical) of a
reference protein having a length of 10, 20, 30, 40, 50, 60, 70,
80, 90, 100 or longer than 100 amino acids. In another example, any
protein that includes a stretch of 20, 30, 40, 50, or 100
(contiguous) amino acids that are 40%, 50%, 60%, 70%, 80%, 90%,
95%, or 100% identical to any of the sequences described herein can
be utilized in accordance with the disclosure. In some embodiments,
a polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
mutations as shown in any of the sequences provided herein or
referenced herein. In another example, any protein that includes a
stretch of 20, 30, 40, 50, or 100 amino acids that are greater than
80%, 90%, 95%, or 100% identical to any of the sequences described
herein, wherein the protein has a stretch of 5, 10, 15, 20, 25, or
30 amino acids that are less than 80%, 75%, 70%, 65% to 60%
identical to any of the sequences described herein can be utilized
in accordance with the disclosure.
[0134] Polypeptide or polynucleotide molecules of the present
disclosure may share a certain degree of sequence similarity or
identity with the reference molecules (e.g., reference polypeptides
or reference polynucleotides), for example, with art-described
molecules (e.g., engineered or designed molecules or wild-type
molecules). The term "identity," as known in the art, refers to a
relationship between the sequences of two or more polypeptides or
polynucleotides, as determined by comparing the sequences. In the
art, identity also means the degree of sequence relatedness between
two sequences as determined by the number of matches between
strings of two or more amino acid residues or nucleic acid
residues. Identity measures the percent of identical matches
between the smaller of two or more sequences with gap alignments
(if any) addressed by a particular mathematical model or computer
program (e.g., "algorithms"). Identity of related peptides can be
readily calculated by known methods. "% identity" as it applies to
polypeptide or polynucleotide sequences is defined as the
percentage of residues (amino acid residues or nucleic acid
residues) in the candidate amino acid or nucleic acid sequence that
are identical with the residues in the amino acid sequence or
nucleic acid sequence of a second sequence after aligning the
sequences and introducing gaps, if necessary, to achieve the
maximum percent identity. Methods and computer programs for the
alignment are well known in the art. Identity depends on a
calculation of percent identity but may differ in value due to gaps
and penalties introduced in the calculation. Generally, variants of
a particular polynucleotide or polypeptide have at least 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to
that particular reference polynucleotide or polypeptide as
determined by sequence alignment programs and parameters described
herein and known to those skilled in the art. Such tools for
alignment include those of the BLAST suite (Stephen F. Altschul, et
al. (1997)." Gapped BLAST and PSI-BLAST: a new generation of
protein database search programs," Nucleic Acids Res.
25:3389-3402). Another popular local alignment technique is based
on the Smith-Waterman algorithm (Smith, T. F. & Waterman, M. S.
(1981) "Identification of common molecular subsequences." J. Mol.
Biol. 147:195-197). A general global alignment technique based on
dynamic programming is the Needleman-Wunsch algorithm (Needleman,
S. B. & Wunsch, C. D. (1970) "A general method applicable to
the search for similarities in the amino acid sequences of two
proteins." J. Mol. Biol. 48:443-453). More recently, a Fast Optimal
Global Sequence Alignment Algorithm (FOGSAA) was developed that
purportedly produces global alignment of nucleotide and protein
sequences faster than other optimal global alignment methods,
including the Needleman-Wunsch algorithm. Other tools are described
herein, specifically in the definition of "identity" below.
[0135] As used herein, the term "homology" refers to the overall
relatedness between polymeric molecules, e.g. between nucleic acid
molecules (e.g. DNA molecules and/or RNA molecules) and/or between
polypeptide molecules. Polymeric molecules (e.g. nucleic acid
molecules (e.g. DNA molecules and/or RNA molecules) and/or
polypeptide molecules) that share a threshold level of similarity
or identity determined by alignment of matching residues are termed
homologous. Homology is a qualitative term that describes a
relationship between molecules and can be based upon the
quantitative similarity or identity. Similarity or identity is a
quantitative term that defines the degree of sequence match between
two compared sequences. In some embodiments, polymeric molecules
are considered to be "homologous" to one another if their sequences
are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, or 99% identical or similar. The term
"homologous" necessarily refers to a comparison between at least
two sequences (polynucleotide or polypeptide sequences). Two
polynucleotide sequences are considered homologous if the
polypeptides they encode are at least 50%, 60%, 70%, 80%, 90%, 95%,
or even 99% for at least one stretch of at least 20 amino acids. In
some embodiments, homologous polynucleotide sequences are
characterized by the ability to encode a stretch of at least 4-5
uniquely specified amino acids. For polynucleotide sequences less
than 60 nucleotides in length, homology is determined by the
ability to encode a stretch of at least 4-5 uniquely specified
amino acids. Two protein sequences are considered homologous if the
proteins are at least 50%, 60%, 70%, 80%, or 90% identical for at
least one stretch of at least 20 amino acids.
[0136] Homology implies that the compared sequences diverged in
evolution from a common origin. The term "homolog" refers to a
first amino acid sequence or nucleic acid sequence (e.g., gene (DNA
or RNA) or protein sequence) that is related to a second amino acid
sequence or nucleic acid sequence by descent from a common
ancestral sequence. The term "homolog" may apply to the
relationship between genes and/or proteins separated by the event
of speciation or to the relationship between genes and/or proteins
separated by the event of genetic duplication. "Orthologs" are
genes (or proteins) in different species that evolved from a common
ancestral gene (or protein) by speciation. Typically, orthologs
retain the same function in the course of evolution. "Paralogs" are
genes (or proteins) related by duplication within a genome.
Orthologs retain the same function in the course of evolution,
whereas paralogs evolve new functions, even if these are related to
the original one.
[0137] The term "identity" refers to the overall relatedness
between polymeric molecules, for example, between polynucleotide
molecules (e.g. DNA molecules and/or RNA molecules) and/or between
polypeptide molecules. Calculation of the percent identity of two
polynucleic acid sequences, for example, can be performed by
aligning the two sequences for optimal comparison purposes (e.g.,
gaps can be introduced in one or both of a first and a second
nucleic acid sequences for optimal alignment and non-identical
sequences can be disregarded for comparison purposes). In certain
embodiments, the length of a sequence aligned for comparison
purposes is at least 30%, at least 40%, at least 50%, at least 60%,
at least 70%, at least 80%, at least 90%, at least 95%, or 100% of
the length of the reference sequence. The nucleotides at
corresponding nucleotide positions are then compared. When a
position in the first sequence is occupied by the same nucleotide
as the corresponding position in the second sequence, then the
molecules are identical at that position. The percent identity
between the two sequences is a function of the number of identical
positions shared by the sequences, taking into account the number
of gaps, and the length of each gap, which needs to be introduced
for optimal alignment of the two sequences. The comparison of
sequences and determination of percent identity between two
sequences can be accomplished using a mathematical algorithm. For
example, the percent identity between two nucleic acid sequences
can be determined using methods such as those described in
Computational Molecular Biology, Lesk, A. M., ed., Oxford
University Press, New York, 1988; Biocomputing: Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Sequence Analysis in Molecular Biology, von Heinje, G., Academic
Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin,
A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994;
and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds.,
M Stockton Press, New York, 1991; each of which is incorporated
herein by reference. For example, the percent identity between two
nucleic acid sequences can be determined using the algorithm of
Meyers and Miller (CABIOS, 1989, 4:11-17), which has been
incorporated into the ALIGN program (version 2.0) using a PAM120
weight residue table, a gap length penalty of 12 and a gap penalty
of 4. The percent identity between two nucleic acid sequences can,
alternatively, be determined using the GAP program in the GCG
software package using an NWSgapdna.CMP matrix. Methods commonly
employed to determine percent identity between sequences include,
but are not limited to those disclosed in Carillo, H., and Lipman,
D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by
reference. Techniques for determining identity are codified in
publicly available computer programs. Exemplary computer software
to determine homology between two sequences include, but are not
limited to, GCG program package, Devereux, J., et al., Nucleic
Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA
Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990)).
Multiprotein and Multicomponent Vaccines
[0138] The present disclosure encompasses bacterial vaccines
comprising multiple RNA (e.g., mRNA) polynucleotides, each encoding
a single antigenic polypeptide, as well as bacterial vaccines
comprising a single RNA polynucleotide encoding more than one
antigenic polypeptide (e.g., as a fusion polypeptide). Thus, a
vaccine composition comprising a RNA (e.g., mRNA) polynucleotide
having an open reading frame encoding a first antigenic polypeptide
and a RNA (e.g., mRNA) polynucleotide having an open reading frame
encoding a second antigenic polypeptide encompasses (a) vaccines
that comprise a first RNA polynucleotide encoding a first antigenic
polypeptide and a second RNA polynucleotide encoding a second
antigenic polypeptide, and (b) vaccines that comprise a single RNA
polynucleotide encoding a first and second antigenic polypeptide
(e.g., as a fusion polypeptide). RNA (e.g., mRNA) vaccines of the
present disclosure, in some embodiments, comprise 2-10 (e.g., 2, 3,
4, 5, 6, 7, 8, 9 or 10), or more, RNA polynucleotides having an
open reading frame, each of which encodes a different antigenic
polypeptide (or a single RNA polynucleotide encoding 2-10, or more,
different antigenic polypeptides). The antigenic polypeptides may
be selected from bacterial antigenic polypeptides.
[0139] In some embodiments, a multicomponent vaccine comprises at
least one RNA (e.g., mRNA) polynucleotide encoding at least one
antigenic polypeptide fused to a signal peptide (e.g., any one of
SEQ ID NO: 39-43). The signal peptide may be fused at the
N-terminus or the C-terminus of an antigenic polypeptide.
Signal Peptides
[0140] In some embodiments, antigenic polypeptides encoded by
bacterial RNA (e.g., mRNA) polynucleotides comprise a signal
peptide. Signal peptides, comprising the N-terminal 15-60 amino
acids of proteins, are typically needed for the translocation
across the membrane on the secretory pathway and, thus, universally
control the entry of most proteins both in eukaryotes and
prokaryotes to the secretory pathway. Signal peptides generally
include three regions: an N-terminal region of differing length,
which usually comprises positively charged amino acids; a
hydrophobic region; and a short carboxy-terminal peptide region. In
eukaryotes, the signal peptide of a nascent precursor protein
(pre-protein) directs the ribosome to the rough endoplasmic
reticulum (ER) membrane and initiates the transport of the growing
peptide chain across it for processing. ER processing produces
mature proteins, wherein the signal peptide is cleaved from
precursor proteins, typically by an ER-resident signal peptidase of
the host cell, or they remain uncleaved and function as a membrane
anchor. A signal peptide may also facilitate the targeting of the
protein to the cell membrane. The signal peptide, however, is not
responsible for the final destination of the mature protein.
Secretory proteins devoid of additional address tags in their
sequence are by default secreted to the external environment.
During recent years, a more advanced view of signal peptides has
evolved, showing that the functions and immunodominance of certain
signal peptides are much more versatile than previously
anticipated.
[0141] A signal peptide may have a length of 15-60 amino acids. For
example, a signal peptide may have a length of 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, or 60 amino acids. In some embodiments, a
signal peptide has a length of 20-60, 25-60, 30-60, 35-60, 40-60,
45-60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55,
45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50,
15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20-40, 25-40,
30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30,
15-25, 20-25, or 15-20 amino acids.
[0142] Bacterial vaccines of the present disclosure may comprise,
for example, RNA (e.g., mRNA) polynucleotides encoding an
artificial signal peptide, wherein the signal peptide coding
sequence is operably linked to and is in frame with the coding
sequence of the antigenic polypeptide. Thus, bacterial vaccines of
the present disclosure, in some embodiments, produce an antigenic
polypeptide comprising an antigenic polypeptide fused to a signal
peptide. In some embodiments, a signal peptide is fused to the
N-terminus of the antigenic polypeptide. In some embodiments, a
signal peptide is fused to the C-terminus of the antigenic
polypeptide.
[0143] In some embodiments, the signal peptide fused to the
antigenic polypeptide is an artificial signal peptide. In some
embodiments, an artificial signal peptide fused to the antigenic
polypeptide encoded by the RNA (e.g., mRNA) vaccine is obtained
from an immunoglobulin protein, e.g., an IgE signal peptide or an
IgG signal peptide. In some embodiments, a signal peptide fused to
the antigenic polypeptide encoded by a RNA (e.g., mRNA) vaccine is
an Ig heavy chain epsilon-1 signal peptide (IgE HC SP) having the
sequence of: MDWTWILFLVAAATRVHS (SEQ ID NO: 40). In some
embodiments, a signal peptide fused to the antigenic polypeptide
encoded by the (e.g., mRNA) RNA (e.g., mRNA) vaccine is an IgGk
chain V-III region HAH signal peptide (IgGk SP) having the sequence
of METPAQLLFLLLLWLPDTTG (SEQ ID NO: 39). In some embodiments, the
signal peptide is selected from: Japanese encephalitis PRM signal
sequence (MLGSNSGQRVVFTILLLLVAPAYS; SEQ ID NO: 41), VSVg protein
signal sequence (MKCLLYLAFLFIGVNCA; SEQ ID NO: 42) and Japanese
encephalitis JEV signal sequence (MWLVSLAIVTACAGA; SEQ ID NO:
43).
[0144] In some embodiments, the antigenic polypeptide encoded by a
RNA (e.g., mRNA) vaccine comprises an amino acid sequence
identified by any one of SEQ ID NO: 10-29 (Table 2) fused to a
signal peptide identified by any one of SEQ ID NO: 39-43. The
examples disclosed herein are not meant to be limiting and any
signal peptide that is known in the art to facilitate targeting of
a protein to ER for processing and/or targeting of a protein to the
cell membrane may be used in accordance with the present
disclosure.
[0145] A signal peptide may have a length of 15-60 amino acids. For
example, a signal peptide may have a length of 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,
37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 55, 56, 57, 58, 59, or 60 amino acids. In some embodiments, a
signal peptide has a length of 20-60, 25-60, 30-60, 35-60, 40-60,
45-60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55,
45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50,
15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20-40, 25-40,
30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30,
15-25, 20-25, or 15-20 amino acids.
[0146] A signal peptide is typically cleaved from the nascent
polypeptide at the cleavage junction during ER processing. The
mature antigenic polypeptide produce by a bacterial RNA (e.g.,
mRNA) vaccine of the present disclosure typically does not comprise
a signal peptide.
Fusion Proteins
[0147] In some embodiments, a bacterial RNA vaccine of the present
disclosure includes an RNA encoding an antigenic fusion protein.
Thus, the encoded antigen or antigens may include two or more
proteins (e.g., protein and/or protein fragment) joined together.
Alternatively, the protein to which a protein antigen is fused does
not promote a strong immune response to itself, but rather to the
antigen. Antigenic fusion proteins, in some embodiments, retain the
functional property from each original protein.
Scaffold Moieties
[0148] The RNA (e.g., mRNA) vaccines as provided herein, in some
embodiments, encode fusion proteins which comprise bacterial
antigens linked to scaffold moieties. In some embodiments, such
scaffold moieties impart desired properties to an antigen encoded
by a nucleic acid of the disclosure. For example scaffold proteins
may improve the immunogenicity of an antigen, e.g., by altering the
structure of the antigen, altering the uptake and processing of the
antigen, and/or causing the antigen to bind to a binding
partner.
[0149] In some embodiments, the scaffold moiety is protein that can
self-assemble into protein nanoparticles that are highly symmetric,
stable, and structurally organized, with diameters of 10-150 nm, a
highly suitable size range for optimal interactions with various
cells of the immune system. In some embodiments, viral proteins or
virus-like particles can be used to form stable nanoparticle
structures. Examples of such viral proteins are known in the art.
For example, in some embodiments, the scaffold moiety is a
hepatitis B surface antigen (HBsAg). HBsAg forms spherical
particles with an average diameter of .about.22 nm and which lacked
nucleic acid and hence are non-infectious (Lopez-Sagaseta, J. et
al. Computational and Structural Biotechnology Journal 14 (2016)
58-68). In some embodiments, the scaffold moiety is a hepatitis B
core antigen (HBcAg) self-assembles into particles of 24-31 nm
diameter, which resembled the viral cores obtained from
HBV-infected human liver. HBcAg produced in self-assembles into two
classes of differently sized nanoparticles of 300 .ANG. and 360
.ANG. diameter, corresponding to 180 or 240 protomers. In some
embodiments a bacterial antigen is fused to HBsAG or HBcAG to
facilitate self-assembly of nanoparticles displaying the bacterial
antigen.
[0150] In another embodiment, bacterial protein platforms may be
used. Non-limiting examples of these self-assembling proteins
include ferritin, lumazine and encapsulin.
[0151] Ferritin is a protein whose main function is intracellular
iron storage. Ferritin is made of 24 subunits, each composed of a
four-alpha-helix bundle, that self-assemble in a quaternary
structure with octahedral symmetry (Cho K. J. et al. J Mol Biol.
2009; 390:83-98). Several high-resolution structures of ferritin
have been determined, confirming that Helicobacter pylori ferritin
is made of 24 identical protomers, whereas in animals, there are
ferritin light and heavy chains that can assemble alone or combine
with different ratios into particles of 24 subunits (Granier T. et
al. J Biol Inorg Chem. 2003; 8:105-111; Lawson D. M. et al. Nature.
1991; 349:541-544). Ferritin self-assembles into nanoparticles with
robust thermal and chemical stability. Thus, the ferritin
nanoparticle is well-suited to carry and expose antigens.
[0152] Lumazine synthase (LS) is also well-suited as a nanoparticle
platform for antigen display. LS, which is responsible for the
penultimate catalytic step in the biosynthesis of riboflavin, is an
enzyme present in a broad variety of organisms, including archaea,
bacteria, fungi, plants, and eubacteria (Weber S. E. Flavins and
Flavoproteins. Methods and Protocols, Series: Methods in Molecular
Biology. 2014). The LS monomer is 150 amino acids long, and
consists of beta-sheets along with tandem alpha-helices flanking
its sides. A number of different quaternary structures have been
reported for LS, illustrating its morphological versatility: from
homopentamers up to symmetrical assemblies of 12 pentamers forming
capsids of 150 .ANG. diameter. Even LS cages of more than 100
subunits have been described (Zhang X. et al. J Mol Biol. 2006;
362:753-770).
[0153] Encapsulin, a novel protein cage nanoparticle isolated from
thermophile Thermotoga maritima, may also be used as a platform to
present antigens on the surface of self-assembling nanoparticles.
Encapsulin is assembled from 60 copies of identical 31 kDa monomers
having a thin and icosahedral T=1 symmetric cage structure with
interior and exterior diameters of 20 and 24 nm, respectively
(Sutter M. et al. Nat Struct Mol Biol. 2008, 15: 939-947). Although
the exact function of encapsulin in T. maritima is not clearly
understood yet, its crystal structure has been recently solved and
its function was postulated as a cellular compartment that
encapsulates proteins such as DyP (Dye decolorizing peroxidase) and
Flp (Ferritin like protein), which are involved in oxidative stress
responses (Rahmanpour R. et al. FEBS J. 2013, 280: 2097-2104).
Linkers and Cleavable Peptides
[0154] In some embodiments, the mRNAs of the disclosure encode more
than one polypeptide, referred to herein as fusion proteins. In
some embodiments, the mRNA further encodes a linker located between
at least one or each domain of the fusion protein. The linker can
be, for example, a cleavable linker or protease-sensitive linker.
In some embodiments, the linker is selected from the group
consisting of F2A linker, P2A linker, T2A linker, E2A linker, and
combinations thereof. This family of self-cleaving peptide linkers,
referred to as 2 .ANG. peptides, has been described in the art (see
for example, Kim, J. H. et al. (2011) PLoS ONE 6:e18556). In some
embodiments, the linker is an F2A linker. In some embodiments, the
linker is a GGGS linker. In some embodiments, the fusion protein
contains three domains with intervening linkers, having the
structure: domain-linker-domain-linker-domain. Cleavable linkers
known in the art may be used in connection with the disclosure.
Exemplary such linkers include: F2A linkers, T2A linkers, P2A
linkers, E2A linkers (See, e.g., WO2017127750). The skilled artisan
will appreciate that other art-recognized linkers may be suitable
for use in the constructs of the disclosure (e.g., encoded by the
nucleic acids of the disclosure). The skilled artisan will likewise
appreciate that other polycistronic constructs (mRNA encoding more
than one antigen/polypeptide separately within the same molecule)
may be suitable for use as provided herein.
Chemically Unmodified Nucleotides
[0155] In some embodiments, at least one RNA (e.g., mRNA) of a
bacterial vaccine of the present disclosure is not chemically
modified and comprises the standard ribonucleotides consisting of
adenosine, guanosine, cytosine and uridine. In some embodiments,
nucleotides and nucleosides of the present disclosure comprise
standard nucleoside residues such as those present in transcribed
RNA (e.g. A, G, C, or U). In some embodiments, nucleotides and
nucleosides of the present disclosure comprise standard
deoxyribonucleosides such as those present in DNA (e.g. dA, dG, dC,
or dT).
Chemical Modifications
[0156] Bacterial vaccines of the present disclosure, in some
embodiments, comprise at least RNA (e.g. mRNA) polynucleotide
having an open reading frame encoding at least one antigenic
polypeptide, wherein the nucleic acid comprises nucleotides and/or
nucleosides that can be standard (unmodified) or modified as is
known in the art. In some embodiments, nucleotides and nucleosides
of the present disclosure comprise modified nucleotides or
nucleosides. Such modified nucleotides and nucleosides can be
naturally-occurring modified nucleotides and nucleosides or
non-naturally occurring modified nucleotides and nucleosides. Such
modifications can include those at the sugar, backbone, or
nucleobase portion of the nucleotide and/or nucleoside as are
recognized in the art.
[0157] The terms "chemical modification" and "chemically modified"
refer to modification with respect to adenosine (A), guanosine (G),
uridine (U), thymidine (T) or cytidine (C) ribonucleosides or
deoxyribnucleosides in at least one of their position, pattern,
percent or population. Generally, these terms do not refer to the
ribonucleotide modifications in naturally occurring 5'-terminal
mRNA cap moieties. With respect to a polypeptide, the term
"modification" refers to a modification relative to the canonical
set 20 amino acids. Polypeptides, as provided herein, are also
considered "modified" of they contain amino acid substitutions,
insertions or a combination of substitutions and insertions.
[0158] Polynucleotides (e.g., RNA polynucleotides, such as mRNA
polynucleotides), in some embodiments, comprise various (more than
one) different modifications. In some embodiments, a particular
region of a polynucleotide contains one, two or more (optionally
different) nucleoside or nucleotide modifications. In some
embodiments, a modified RNA polynucleotide (e.g., a modified mRNA
polynucleotide), introduced to a cell or organism, exhibits reduced
degradation in the cell or organism, respectively, relative to an
unmodified polynucleotide. In some embodiments, a modified RNA
polynucleotide (e.g., a modified mRNA polynucleotide), introduced
into a cell or organism, may exhibit reduced immunogenicity in the
cell or organism, respectively (e.g., a reduced innate
response).
[0159] Modifications of polynucleotides include, without
limitation, those described herein. Polynucleotides (e.g., RNA
polynucleotides, such as mRNA polynucleotides) may comprise
modifications that are naturally-occurring, non-naturally-occurring
or the polynucleotide may comprise a combination of
naturally-occurring and non-naturally-occurring modifications.
Polynucleotides may include any useful modification, for example,
of a sugar, a nucleobase, or an internucleoside linkage (e.g., to a
linking phosphate, to a phosphodiester linkage or to the
phosphodiester backbone).
[0160] Polynucleotides (e.g., RNA polynucleotides, such as mRNA
polynucleotides), in some embodiments, comprise non-natural
modified nucleotides that are introduced during synthesis or
post-synthesis of the polynucleotides to achieve desired functions
or properties. The modifications may be present on an
internucleotide linkages, purine or pyrimidine bases, or sugars.
The modification may be introduced with chemical synthesis or with
a polymerase enzyme at the terminal of a chain or anywhere else in
the chain. Any of the regions of a polynucleotide may be chemically
modified.
[0161] The present disclosure provides for modified nucleosides and
nucleotides of a polynucleotide (e.g., RNA polynucleotides, such as
mRNA polynucleotides). A "nucleoside" refers to a compound
containing a sugar molecule (e.g., a pentose or ribose) or a
derivative thereof in combination with an organic base (e.g., a
purine or pyrimidine) or a derivative thereof (also referred to
herein as "nucleobase"). A nucleotide" refers to a nucleoside,
including a phosphate group. Modified nucleotides may by
synthesized by any useful method, such as, for example, chemically,
enzymatically, or recombinantly, to include one or more modified or
non-natural nucleosides. Polynucleotides may comprise a region or
regions of linked nucleosides. Such regions may have variable
backbone linkages. The linkages may be standard phosphdioester
linkages, in which case the polynucleotides would comprise regions
of nucleotides.
[0162] Modified nucleotide base pairing encompasses not only the
standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine
base pairs, but also base pairs formed between nucleotides and/or
modified nucleotides comprising non-standard or modified bases,
wherein the arrangement of hydrogen bond donors and hydrogen bond
acceptors permits hydrogen bonding between a non-standard base and
a standard base or between two complementary non-standard base
structures. One example of such non-standard base pairing is the
base pairing between the modified nucleotide inosine and adenine,
cytosine or uracil. Any combination of base/sugar or linker may be
incorporated into polynucleotides of the present disclosure.
[0163] Modifications of polynucleotides (e.g., RNA polynucleotides,
such as mRNA polynucleotides) that are useful in the vaccines of
the present disclosure include, but are not limited to the
following: 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine;
2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonyl
carbamoyladenosine; N6-glycinylcarbamoyladenosine;
N6-isopentenyladenosine; N6-methyladenosine;
N6-threonylcarbamoyladeno sine; 1,2'-O-dimethyladenosine;
1-methyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine
(phosphate); 2-methyladenosine; 2-methylthio-N6
isopentenyladenosine; 2-methylthio-N6-hydroxynorvalyl
carbamoyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine
(phosphate); Isopentenyladenosine;
N6-(cis-hydroxyisopentenyl)adenosine; N6,2'-O-dimethyladenosine;
N6,2'-O-dimethyladenosine; N6,N6,2'-O-trimethyladenosine;
N6,N6-dimethyladenosine; N6-acetyladenosine;
N6-hydroxynorvalylcarbamoyladenosine;
N6-methyl-N6-threonylcarbamoyladenosine; 2-methyladenosine;
2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine;
N1-methyl-adenosine; N6, N6 (dimethyl)adenine;
N6-cis-hydroxy-isopentenyl-adenosine; .alpha.-thio-adenosine; 2
(amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6
(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine;
2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine;
2-(propyl)adenine; 2'-Amino-2'-deoxy-ATP; 2'-Azido-2'-deoxy-ATP;
2'-Deoxy-2'-a-aminoadenosine TP; 2'-Deoxy-2'-a-azidoadenosine TP; 6
(alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine;
6-(methyl)adenine; 7 (deaza)adenine; 8 (alkenyl)adenine; 8
(alkynyl)adenine; 8 (amino)adenine; 8 (thioalkyl)adenine;
8-(alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine;
8-(amino)adenine; 8-(halo)adenine; 8-(hydroxyl)adenine;
8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azido-adeno sine; aza
adenine; deaza adenine; N6 (methyl)adenine; N6-(isopentyl)adenine;
7-deaza-8-aza-adenosine; 7-methyladenine; 1-Deazaadenosine TP;
2'Fluoro-N6-Bz-deoxyadenosine TP; 2'-OMe-2-Amino-ATP;
2'O-methyl-N6-Bz-deoxyadenosine TP; 2'-a-Ethynyladenosine TP;
2-aminoadenine; 2-Aminoadenosine TP; 2-Amino-ATP;
2'-a-Trifluoromethyladenosine TP; 2-Azidoadenosine TP;
2'-b-Ethynyladenosine TP; 2-Bromoadenosine TP;
2'-b-Trifluoromethyladenosine TP; 2-Chloroadenosine TP;
2'-Deoxy-2',2'-difluoroadenosine TP;
2'-Deoxy-2'-a-mercaptoadenosine TP;
2'-Deoxy-2'-a-thiomethoxyadenosine TP; 2'-Deoxy-2'-b-aminoadenosine
TP; 2'-Deoxy-2'-b-azidoadenosine TP; 2'-Deoxy-2'-b-bromoadenosine
TP; 2'-Deoxy-2'-b-chloroadenosine TP; 2'-Deoxy-2'-b-fluoroadenosine
TP; 2'-Deoxy-2'-b-iodoadenosine TP; 2'-Deoxy-2'-b-mercaptoadenosine
TP; 2'-Deoxy-2'-b-thiomethoxyadenosine TP; 2-Fluoroadenosine TP;
2-Iodoadenosine TP; 2-Mercaptoadenosine TP; 2-methoxy-adenine;
2-methylthio-adenine; 2-Trifluoromethyladenosine TP;
3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP;
3-Deaza-3-fluoroadenosine TP; 3-Deaza-3-iodoadenosine TP;
3-Deazaadenosine TP; 4'-Azidoadenosine TP; 4'-Carbocyclic adenosine
TP; 4'-Ethynyladenosine TP; 5'-Homo-adenosine TP; 8-Aza-ATP;
8-bromo-adenosine TP; 8-Trifluoromethyladenosine TP;
9-Deazaadenosine TP; 2-aminopurine; 7-deaza-2,6-diaminopurine;
7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine;
2,6-diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine;
2-thiocytidine; 3-methylcytidine; 5-formylcytidine;
5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine;
2'-O-methylcytidine; 2'-O-methylcytidine; 5,2'-O-dimethylcytidine;
5-formyl-2'-O-methylcytidine; Lysidine; N4,2'-O-dimethylcytidine;
N4-acetyl-2'-O-methylcytidine; N4-methylcytidine;
N4,N4-Dimethyl-2'-OMe-Cytidine TP; 4-methylcytidine;
5-aza-cytidine; Pseudo-iso-cytidine; pyrrolo-cytidine;
.alpha.-thio-cytidine; 2-(thio)cytosine; 2'-Amino-2'-deoxy-CTP;
2'-Azido-2'-deoxy-CTP; 2'-Deoxy-2'-a-aminocytidine TP;
2'-Deoxy-2'-a-azidocytidine TP; 3 (deaza) 5 (aza)cytosine; 3
(methyl)cytosine; 3-(alkyl)cytosine; 3-(deaza) 5 (aza)cytosine;
3-(methyl)cytidine; 4,2'-O-dimethylcytidine; 5 (halo)cytosine; 5
(methyl)cytosine; 5 (propynyl)cytosine; 5
(trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine;
5-(halo)cytosine; 5-(propynyl)cytosine;
5-(trifluoromethyl)cytosine; 5-bromo-cytidine; 5-iodo-cytidine;
5-propynyl cytosine; 6-(azo)cytosine; 6-aza-cytidine; aza cytosine;
deaza cytosine; N4 (acetyl)cytosine;
1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine;
2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine;
2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine;
4-methoxy-pseudoisocytidine;
4-thio-1-methyl-1-deaza-pseudoisocytidine;
4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine;
5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine;
Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2'-anhydro-cytidine
TP hydrochloride; 2'Fluor-N4-Bz-cytidine TP;
2'Fluoro-N4-Acetyl-cytidine TP; 2'-O-Methyl-N4-Acetyl-cytidine TP;
2'O-methyl-N4-Bz-cytidine TP; 2'-a-Ethynylcytidine TP;
2'-a-Trifluoromethylcytidine TP; 2'-b-Ethynylcytidine TP;
2'-b-Trifluoromethylcytidine TP; 2'-Deoxy-2',2'-difluorocytidine
TP; 2'-Deoxy-2'-a-mercaptocytidine TP;
2'-Deoxy-2'-a-thiomethoxycytidine TP; 2'-Deoxy-2'-b-aminocytidine
TP; 2'-Deoxy-2'-b-azidocytidine TP; 2'-Deoxy-2'-b-bromocytidine TP;
2'-Deoxy-2'-b-chlorocytidine TP; 2'-Deoxy-2'-b-fluorocytidine TP;
2'-Deoxy-2'-b-iodocytidine TP; 2'-Deoxy-2'-b-mercaptocytidine TP;
2'-Deoxy-2'-b-thiomethoxycytidine TP;
2'-O-Methyl-5-(1-propynyl)cytidine TP; 3'-Ethynylcytidine TP;
4'-Azidocytidine TP; 4'-Carbocyclic cytidine TP; 4'-Ethynylcytidine
TP; 5-(1-Propynyl)ara-cytidine TP;
5-(2-Chloro-phenyl)-2-thiocytidine TP;
5-(4-Amino-phenyl)-2-thiocytidine TP; 5-Aminoallyl-CTP;
5-Cyanocytidine TP; 5-Ethynylara-cytidine TP; 5-Ethynylcytidine TP;
5'-Homo-cytidine TP; 5-Methoxycytidine TP;
5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP;
N4-Benzoyl-cytidine TP; Pseudoisocytidine; 7-methylguanosine;
N2,2'-O-dimethylguanosine; N2-methylguanosine; Wyosine;
1,2'-O-dimethylguanosine; 1-methylguanosine; 2'-O-methylguanosine;
2'-O-ribosylguanosine (phosphate); 2'-O-methylguanosine;
2'-O-ribosylguanosine (phosphate); 7-aminomethyl-7-deazaguanosine;
7-cyano-7-deazaguanosine; Archaeosine; Methylwyosine;
N2,7-dimethylguanosine; N2,N2,2'-O-trimethylguanosine;
N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine;
N2,7,2'-O-trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine;
8-oxo-guanosine; N1-methyl-guanosine; .alpha.-thio-guanosine; 2
(propyl)guanine; 2-(alkyl)guanine; 2'-Amino-2'-deoxy-GTP;
2'-Azido-2'-deoxy-GTP; 2'-Deoxy-2'-a-aminoguanosine TP;
2'-Deoxy-2'-a-azidoguanosine TP; 6 (methyl)guanine;
6-(alkyl)guanine; 6-(methyl)guanine; 6-methyl-guanosine; 7
(alkyl)guanine; 7 (deaza)guanine; 7 (methyl)guanine;
7-(alkyl)guanine; 7-(deaza)guanine; 7-(methyl)guanine; 8
(alkyl)guanine; 8 (alkynyl)guanine; 8 (halo)guanine; 8
(thioalkyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine;
8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine;
8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; aza
guanine; deaza guanine; N (methyl)guanine; N-(methyl)guanine;
1-methyl-6-thio-guanosine; 6-methoxy-guanosine;
6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine;
6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine;
7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine;
N2-methyl-6-thio-guanosine; 1-Me-GTP;
2'Fluoro-N2-isobutyl-guanosine TP; 2'O-methyl-N2-isobutyl-guanosine
TP; 2'-a-Ethynylguanosine TP; 2'-a-Trifluoromethylguanosine TP;
2'-b-Ethynylguanosine TP; 2'-b-Trifluoromethylguanosine TP;
2'-Deoxy-2',2'-difluoroguanosine TP;
2'-Deoxy-2'-a-mercaptoguanosine TP;
2'-Deoxy-2'-a-thiomethoxyguanosine TP; 2'-Deoxy-2'-b-aminoguanosine
TP; 2'-Deoxy-2'-b-azidoguanosine TP; 2'-Deoxy-2'-b-bromoguanosine
TP; 2'-Deoxy-2'-b-chloroguanosine TP; 2'-Deoxy-2'-b-fluoroguanosine
TP; 2'-Deoxy-2'-b-iodoguanosine TP; 2'-Deoxy-2'-b-mercaptoguanosine
TP; 2'-Deoxy-2'-b-thiomethoxyguanosine TP; 4'-Azidoguanosine TP;
4'-Carbocyclic guanosine TP; 4'-Ethynylguanosine TP;
5'-Homo-guanosine TP; 8-bromo-guanosine TP; 9-Deazaguanosine TP;
N2-isobutyl-guanosine TP; 1-methylinosine; Inosine;
1,2'-O-dimethylinosine; 2'-O-methylinosine; 7-methylinosine;
2'-O-methylinosine; Epoxyqueuosine; galactosyl-queuosine;
Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine;
deaza thymidine; deoxy-thymidine; 2'-O-methyluridine;
2-thiouridine; 3-methyluridine; 5-carboxymethyluridine;
5-hydroxyuridine; 5-methyluridine; 5-taurinomethyl-2-thiouridine;
5-taurinomethyluridine; Dihydrouridine; Pseudouridine;
(3-(3-amino-3-carboxypropyl)uridine;
1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine;
1-methylpseduouridine; 1-methyl-pseudouridine; 2'-O-methyluridine;
2'-O-methylpseudouridine; 2'-O-methyluridine;
2-thio-2'-O-methyluridine; 3-(3-amino-3-carboxypropyl)uridine;
3,2'-O-dimethyluridine; 3-Methyl-pseudo-Uridine TP; 4-thiouridine;
5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine
methyl ester; 5,2'-O-dimethyluridine; 5,6-dihydro-uridine;
5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2'-O-methyluridine;
5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine;
5-carboxyhydroxymethyluridine methyl ester;
5-carboxymethylaminomethyl-2'-O-methyluridine;
5-carboxymethylaminomethyl-2-thiouridine;
5-carboxymethylaminomethyl-2-thiouridine;
5-carboxymethylaminomethyluridine;
5-carboxymethylaminomethyluridine; 5-Carbamoylmethyluridine TP;
5-methoxycarbonylmethyl-2'-O-methyluridine;
5-methoxycarbonylmethyl-2-thiouridine;
5-methoxycarbonylmethyluridine; 5-methoxyuridine;
5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine;
5-methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine;
5-Methyldihydrouridine; 5-Oxyacetic acid-Uridine TP; 5-Oxyacetic
acid-methyl ester-Uridine TP; N1-methyl-pseudo-uridine; uridine
5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester;
3-(3-Amino-3-carboxypropyl)-Uridine TP;
5-(iso-Pentenylaminomethyl)-2-thiouridine TP;
5-(iso-Pentenylaminomethyl)-2'-O-methyluridine TP;
5-(iso-Pentenylaminomethyl)uridine TP; 5-propynyl uracil;
.alpha.-thio-uridine; 1
(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil; 1
(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1
(aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil; 1
(aminoalkylaminocarbonylethylenyl)-pseudouracil; 1
(aminocarbonylethylenyl)-2(thio)-pseudouracil; 1
(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1
(aminocarbonylethylenyl)-4 (thio)pseudouracil; 1
(aminocarbonylethylenyl)-pseudouracil; 1 substituted
2(thio)-pseudouracil; 1 substituted 2,4-(dithio)pseudouracil; 1
substituted 4 (thio)pseudouracil; 1 substituted pseudouracil;
1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil;
1-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP;
1-Methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP;
1-Methyl-pseudo-UTP; 2 (thio)pseudouracil; 2' deoxy uridine; 2'
fluorouridine; 2-(thio)uracil; 2,4-(dithio)psuedouracil; 2' methyl,
2'amino, 2'azido, 2'fluro-guanosine; 2'-Amino-2'-deoxy-UTP;
2'-Azido-2'-deoxy-UTP; 2'-Azido-deoxyuridine TP;
2'-O-methylpseudouridine; 2' deoxy uridine; 2' fluorouridine;
2'-Deoxy-2'-a-aminouridine TP; 2'-Deoxy-2'-a-azidouridine TP;
2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4
(thio)pseudouracil; 4-(thio)pseudouracil; 4-(thio)uracil;
4-thiouracil; 5 (1,3-diazole-1-alkyl)uracil; 5
(2-aminopropyl)uracil; 5 (aminoalkyl)uracil; 5
(dimethylaminoalkyl)uracil; 5 (guanidiniumalkyl)uracil; 5
(methoxycarbonylmethyl)-2-(thio)uracil; 5
(methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5
(methyl) 2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5
(methylaminomethyl)-2 (thio)uracil; 5 (methylaminomethyl)-2,4
(dithio)uracil; 5 (methylaminomethyl)-4 (thio)uracil; 5
(propynyl)uracil; 5 (trifluoromethyl)uracil;
5-(2-aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouracil;
5-(alkyl)-2,4 (dithio)pseudouracil; 5-(alkyl)-4 (thio)pseudouracil;
5-(alkyl)pseudouracil; 5-(alkyl)uracil; 5-(alkynyl)uracil;
5-(allylamino)uracil; 5-(cyanoalkyl)uracil;
5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil;
5-(guanidiniumalkyl)uracil; 5-(halo)uracil;
5-(1,3-diazole-1-alkyl)uracil; 5-(methoxy)uracil;
5-(methoxycarbonylmethyl)-2-(thio)uracil;
5-(methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil;
5-(methyl) 2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil;
5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4
(dithio)pseudouracil; 5-(methyl)-4 (thio)pseudouracil;
5-(methyl)pseudouracil; 5-(methylaminomethyl)-2 (thio)uracil;
5-(methylaminomethyl)-2,4(dithio)uracil;
5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil;
5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine;
5-iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil;
6-aza-uridine; allyamino-uracil; aza uracil; deaza uracil; N3
(methyl)uracil; P seudo-UTP-1-2-ethanoic acid; Pseudouracil;
4-Thio-pseudo-UTP; 1-carboxymethyl-pseudouridine;
1-methyl-1-deaza-pseudouridine; 1-propynyl-uridine;
1-taurinomethyl-1-methyl-uridine; 1-taurinomethyl-4-thio-uridine;
1-taurinomethyl-pseudouridine; 2-methoxy-4-thio-pseudouridine;
2-thio-1-methyl-1-deaza-pseudouridine;
2-thio-1-methyl-pseudouridine; 2-thio-5-aza-uridine;
2-thio-dihydropseudouridine; 2-thio-dihydrouridine;
2-thio-pseudouridine; 4-methoxy-2-thio-pseudouridine;
4-methoxy-pseudouridine; 4-thio-1-methyl-pseudouridine;
4-thio-pseudouridine; 5-aza-uridine; Dihydropseudouridine;
(.+-.)1-(2-Hydroxypropyl)pseudouridine TP;
(2R)-1-(2-Hydroxypropyl)pseudouridine TP;
(2S)-1-(2-Hydroxypropyl)pseudouridine TP;
(E)-5-(2-Bromo-vinyl)ara-uridine TP; (E)-5-(2-Bromo-vinyl)uridine
TP; (Z)-5-(2-Bromo-vinyl)ara-uridine TP;
(Z)-5-(2-Bromo-vinyl)uridine TP;
1-(2,2,2-Trifluoroethyl)-pseudo-UTP;
1-(2,2,3,3,3-Pentafluoropropyl)pseudouridine TP;
1-(2,2-Diethoxyethyl)pseudouridine TP;
1-(2,4,6-Trimethylbenzyl)pseudouridine TP;
1-(2,4,6-Trimethyl-benzyl)pseudo-UTP;
1-(2,4,6-Trimethyl-phenyl)pseudo-UTP;
1-(2-Amino-2-carboxyethyl)pseudo-UTP; 1-(2-Amino-ethyl)pseudo-UTP;
1-(2-Hydroxyethyl)pseudouridine TP; 1-(2-Methoxyethyl)pseudouridine
TP; 1-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP;
1-(3,4-Dimethoxybenzyl)pseudouridine TP;
1-(3-Amino-3-carboxypropyl)pseudo-UTP;
1-(3-Amino-propyl)pseudo-UTP;
1-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP;
1-(4-Amino-4-carboxybutyl)pseudo-UTP; 1-(4-Amino-benzyl)pseudo-UTP;
1-(4-Amino-butyl)pseudo-UTP; 1-(4-Amino-phenyl)pseudo-UTP;
1-(4-Azidobenzyl)pseudouridine TP; 1-(4-Bromobenzyl)pseudouridine
TP; 1-(4-Chlorobenzyl)pseudouridine TP;
1-(4-Fluorobenzyl)pseudouridine TP; 1-(4-Iodobenzyl)pseudouridine
TP; 1-(4-Methanesulfonylbenzyl)pseudouridine TP;
1-(4-Methoxybenzyl)pseudouridine TP;
1-(4-Methoxy-benzyl)pseudo-UTP; 1-(4-Methoxy-phenyl)pseudo-UTP;
1-(4-Methylbenzyl)pseudouridine TP; 1-(4-Methyl-benzyl)pseudo-UTP;
1-(4-Nitrobenzyl)pseudouridine TP; 1-(4-Nitro-benzyl)pseudo-UTP;
1(4-Nitro-phenyl)pseudo-UTP; 1-(4-Thiomethoxybenzyl)pseudouridine
TP; 1-(4-Trifluoromethoxybenzyl)pseudouridine TP;
1-(4-Trifluoromethylbenzyl)pseudouridine TP;
1-(5-Amino-pentyl)pseudo-UTP; 1-(6-Amino-hexyl)pseudo-UTP;
1,6-Dimethyl-pseudo-UTP;
1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouri-
dine TP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl} pseudouridine
TP; 1-Acetylpseudouridine TP; 1-Alkyl-6-(1-propynyl)-pseudo-UTP;
1-Alkyl-6-(2-propynyl)-pseudo-UTP; 1-Alkyl-6-allyl-pseudo-UTP;
1-Alkyl-6-ethynyl-pseudo-UTP; 1-Alkyl-6-homoallyl-pseudo-UTP;
1-Alkyl-6-vinyl-pseudo-UTP; 1-Allylpseudouridine TP;
1-Aminomethyl-pseudo-UTP; 1-Benzoylpseudouridine TP;
1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP;
1-Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP;
1-Butyl-pseudo-UTP; 1-Cyanomethylpseudouridine TP;
1-Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo-UTP;
1-Cycloheptylmethyl-pseudo-UTP; 1-Cycloheptyl-pseudo-UTP;
1-Cyclohexylmethyl-pseudo-UTP; 1-Cyclohexyl-pseudo-UTP;
1-Cyclooctylmethyl-pseudo-UTP; 1-Cyclooctyl-pseudo-UTP;
1-Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP;
1-Cyclopropylmethyl-pseudo-UTP; 1-Cyclopropyl-pseudo-UTP;
1-Ethyl-pseudo-UTP; 1-Hexyl-pseudo-UTP; 1-Homoallylpseudouridine
TP; 1-Hydroxymethylpseudouridine TP; 1-iso-propyl-pseudo-UTP;
1-Me-2-thio-pseudo-UTP; 1-Me-4-thio-pseudo-UTP;
1-Me-alpha-thio-pseudo-UTP; 1-Methanesulfonylmethylpseudouridine
TP; 1-Methoxymethylpseudouridine TP;
1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP;
1-Methyl-6-(4-morpholino)-pseudo-UTP;
1-Methyl-6-(4-thiomorpholino)-pseudo-UTP; 1-Methyl-6-(substituted
phenyl)pseudo-UTP; 1-Methyl-6-amino-pseudo-UTP;
1-Methyl-6-azido-pseudo-UTP; 1-Methyl-6-bromo-pseudo-UTP;
1-Methyl-6-butyl-pseudo-UTP; 1-Methyl-6-chloro-pseudo-UTP;
1-Methyl-6-cyano-pseudo-UTP; 1-Methyl-6-dimethylamino-pseudo-UTP;
1-Methyl-6-ethoxy-pseudo-UTP;
1-Methyl-6-ethylcarboxylate-pseudo-UTP;
1-Methyl-6-ethyl-pseudo-UTP; 1-Methyl-6-fluoro-pseudo-UTP;
1-Methyl-6-formyl-pseudo-UTP; 1-Methyl-6-hydroxyamino-pseudo-UTP;
1-Methyl-6-hydroxy-pseudo-UTP; 1-Methyl-6-iodo-pseudo-UTP;
1-Methyl-6-iso-propyl-pseudo-UTP; 1-Methyl-6-methoxy-pseudo-UTP;
1-Methyl-6-methylamino-pseudo-UTP; 1-Methyl-6-phenyl-pseudo-UTP;
1-Methyl-6-propyl-pseudo-UTP; 1-Methyl-6-tert-butyl-pseudo-UTP;
1-Methyl-6-trifluoromethoxy-pseudo-UTP;
1-Methyl-6-trifluoromethyl-pseudo-UTP;
1-Morpholinomethylpseudouridine TP; 1-Pentyl-pseudo-UTP;
1-Phenyl-pseudo-UTP; 1-Pivaloylpseudouridine TP;
1-Propargylpseudouridine TP; 1-Propyl-pseudo-UTP;
1-propynyl-pseudouridine; 1-p-tolyl-pseudo-UTP;
1-tert-Butyl-pseudo-UTP; 1-Thiomethoxymethylpseudouridine TP;
1-Thiomorpholinomethylpseudouridine TP;
1-Trifluoroacetylpseudouridine TP; 1-Trifluoromethyl-pseudo-UTP;
1-Vinylpseudouridine TP; 2,2'-anhydro-uridine TP;
2'-bromo-deoxyuridine TP; 2'-F-5-Methyl-2'-deoxy-UTP;
2'-OMe-5-Me-UTP; 2'-OMe-pseudo-UTP; 2'-a-Ethynyluridine TP;
2'-a-Trifluoromethyluridine TP; 2'-b-Ethynyluridine TP;
2'-b-Trifluoromethyluridine TP; 2'-Deoxy-2',2'-difluorouridine TP;
2'-Deoxy-2'-a-mercaptouridine TP; 2'-Deoxy-2'-a-thiomethoxyuridine
TP; 2'-Deoxy-2'-b-aminouridine TP; 2'-Deoxy-2'-b-azidouridine TP;
2'-Deoxy-2'-b-bromouridine TP; 2'-Deoxy-2'-b-chlorouridine TP;
2'-Deoxy-2'-b-fluorouridine TP; 2'-Deoxy-2'-b-iodouridine TP;
2'-Deoxy-2'-b-mercaptouridine TP; 2'-Deoxy-2'-b-thiomethoxyuridine
TP; 2-methoxy-4-thio-uridine; 2-methoxyuridine;
2'-O-Methyl-5-(1-propynyl)uridine TP; 3-Alkyl-pseudo-UTP;
4'-Azidouridine TP; 4'-Carbocyclic uridine TP; 4'-Ethynyluridine
TP; 5-(1-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridine TP;
5-Cyanouridine TP; 5-Dimethylaminouridine TP; 5'-Homo-uridine TP;
5-iodo-2'-fluoro-deoxyuridine TP; 5-Phenylethynyluridine TP;
5-Trideuteromethyl-6-deuterouridine TP; 5-Trifluoromethyl-Uridine
TP; 5-Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP;
6-(4-Morpholino)-pseudo-UTP; 6-(4-Thiomorpholino)-pseudo-UTP;
6-(Substituted-Phenyl)-pseudo-UTP; 6-Amino-pseudo-UTP;
6-Azido-pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl-pseudo-UTP;
6-Chloro-pseudo-UTP; 6-Cyano-pseudo-UTP;
6-Dimethylamino-pseudo-UTP; 6-Ethoxy-pseudo-UTP;
6-Ethylcarboxylate-pseudo-UTP; 6-Ethyl-pseudo-UTP;
6-Fluoro-pseudo-UTP; 6-Formyl-pseudo-UTP;
6-Hydroxyamino-pseudo-UTP; 6-Hydroxy-pseudo-UTP; 6-Iodo-pseudo-UTP;
6-iso-Propyl-pseudo-UTP; 6-Methoxy-pseudo-UTP;
6-Methylamino-pseudo-UTP; 6-Methyl-pseudo-UTP; 6-Phenyl-pseudo-UTP;
6-Phenyl-pseudo-UTP; 6-Propyl-pseudo-UTP; 6-tert-Butyl-pseudo-UTP;
6-Trifluoromethoxy-pseudo-UTP; 6-Trifluoromethyl-pseudo-UTP;
Alpha-thio-pseudo-UTP; Pseudouridine 1-(4-methylbenzenesulfonic
acid) TP; Pseudouridine 1-(4-methylbenzoic acid) TP; Pseudouridine
TP 1-[3-(2-ethoxy)]propionic acid; Pseudouridine TP
1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid;
Pseudouridine TP
1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionic
acid; Pseudouridine TP
1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine
TP 1-[3-{2-(2-ethoxy)-ethoxy}] propionic acid; Pseudouridine TP
1-methylphosphonic acid; Pseudouridine TP 1-methylphosphonic acid
diethyl ester; Pseudo-UTP-N1-3-propionic acid;
Pseudo-UTP-N1-4-butanoic acid; Pseudo-UTP-N1-5-pentanoic acid;
Pseudo-UTP-N1-6-hexanoic acid; Pseudo-UTP-N1-7-heptanoic acid;
Pseudo-UTP-N1-methyl-p-benzoic acid; Pseudo-UTP-N1-p-benzoic acid;
Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine;
undermodified hydroxywybutosine; 4-demethylwyosine;
2,6-(diamino)purine; 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl:
1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;
1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;
1,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2 (amino)purine;
2,4,5-(trimethyl)phenyl; 2' methyl, 2'amino, 2'azido,
2'fluro-cytidine; 2' methyl, 2'amino, 2'azido, 2'fluro-adenine;
2'methyl, 2'amino, 2'azido, 2'fluro-uridine;
2'-amino-2'-deoxyribose; 2-amino-6-Chloro-purine; 2-aza-inosinyl;
2'-azido-2'-deoxyribose; 2'fluoro-2'-deoxyribose;
2'-fluoro-modified bases; 2'-O-methyl-ribose;
2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidine-3-yl;
2-pyridinone; 3 nitropyrrole;
3-(methyl)-7-(propynyl)isocarbostyrilyl;
3-(methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole;
4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl;
5 nitroindole; 5 substituted pyrimidines;
5-(methyl)isocarbostyrilyl; 5-nitroindole; 6-(aza)pyrimidine;
6-(azo)thymine; 6-(methyl)-7-(aza)indolyl; 6-chloro-purine;
6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;
7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;
7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;
7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;
7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;
7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;
7-(aza)indolyl;
7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinl-yl;
7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;
7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;
7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;
7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;
7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;
7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl,
propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted
1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted
1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidizopyridinyl;
Aminoindolyl; Anthracenyl;
bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;
bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;
Difluorotolyl; Hypoxanthine; Imidizopyridinyl; Inosinyl;
Isocarbostyrilyl; Isoguanisine; N2-substituted purines;
N6-methyl-2-amino-purine; N6-substituted purines; N-alkylated
derivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl;
Nitroindazolyl; Nitropyrazolyl; Nubularine; 06-substituted purines;
O-alkylated derivative;
ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;
ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin
TP; para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;
para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Pentacenyl;
Phenanthracenyl; Phenyl; propynyl-7-(aza)indolyl; Pyrenyl;
pyridopyrimidin-3-yl; pyridopyrimidin-3-yl,
2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl;
Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted
1,2,4-triazoles; Tetracenyl; Tubercidine; Xanthine;
Xanthosine-5'-TP; 2-thio-zebularine; 5-aza-2-thio-zebularine;
7-deaza-2-amino-purine; pyridin-4-one ribonucleoside;
2-Amino-riboside-TP; Formycin A TP; Formycin B TP; Pyrrolosine TP;
2'-OH-ara-adenosine TP; 2'-OH-ara-cytidine TP; 2'-OH-ara-uridine
TP; 2'-OH-ara-guanosine TP; 5-(2-carbomethoxyvinyl)uridine TP; and
N6-(19-Amino-pentaoxanonadecyl)adenosine TP.
[0164] In some embodiments, polynucleotides (e.g., RNA
polynucleotides, such as mRNA polynucleotides) include a
combination of at least two (e.g., 2, 3, 4 or more) of the
aforementioned modified nucleobases.
[0165] In some embodiments, modified nucleobases in polynucleotides
(e.g., RNA polynucleotides, such as mRNA polynucleotides) are
selected from the group consisting of pseudouridine (.psi.),
N1-methylpseudouridine (m.sup.1.psi.), 2-thiouridine,
4'-thiouridine, 5-methylcytosine,
2-thio-1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,
2-thio-dihydropseudouridine, 2-thio-dihydrouridine,
2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,
4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,
4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,
5-methoxyuridine and 2'-O-methyl uridine. In some embodiments,
polynucleotides (e.g., RNA polynucleotides, such as mRNA
polynucleotides) include a combination of at least two (e.g., 2, 3,
4 or more) of the aforementioned modified nucleobases.
[0166] In some embodiments, modified nucleobases in polynucleotides
(e.g., RNA polynucleotides, such as mRNA polynucleotides) are
selected from the group consisting of 1-methyl-pseudouridine
(m.sup.1.psi.), 5-methoxy-uridine (mo.sup.5U), 5-methyl-cytidine
(m.sup.5C), pseudouridine (.psi.), .alpha.-thio-guanosine and
.alpha.-thio-adenosine. In some embodiments, polynucleotides
includes a combination of at least two (e.g., 2, 3, 4 or more) of
the aforementioned modified nucleobases.
[0167] In some embodiments, polynucleotides (e.g., RNA
polynucleotides, such as mRNA polynucleotides) comprise
pseudouridine (.psi.) and 5-methyl-cytidine (m.sup.5C). In some
embodiments, polynucleotides (e.g., RNA polynucleotides, such as
mRNA polynucleotides) comprise 1-methyl-pseudouridine
(m.sup.1.psi.). In some embodiments, polynucleotides (e.g., RNA
polynucleotides, such as mRNA polynucleotides) comprise
1-methyl-pseudouridine (m.sup.1.psi.) and 5-methyl-cytidine
(m.sup.5C). In some embodiments, polynucleotides (e.g., RNA
polynucleotides, such as mRNA polynucleotides) comprise
2-thiouridine (s.sup.2U). In some embodiments, polynucleotides
(e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise
2-thiouridine and 5-methyl-cytidine (m.sup.5C). In some
embodiments, polynucleotides (e.g., RNA polynucleotides, such as
mRNA polynucleotides) comprise methoxy-uridine (mo.sup.5U). In some
embodiments, polynucleotides (e.g., RNA polynucleotides, such as
mRNA polynucleotides) comprise 5-methoxy-uridine (mo.sup.5U) and
5-methyl-cytidine (m.sup.5C). In some embodiments, polynucleotides
(e.g., RNA polynucleotides, such as mRNA polynucleotides) comprise
2'-O-methyl uridine. In some embodiments polynucleotides (e.g., RNA
polynucleotides, such as mRNA polynucleotides) comprise 2'-O-methyl
uridine and 5-methyl-cytidine (m.sup.5C). In some embodiments,
polynucleotides (e.g., RNA polynucleotides, such as mRNA
polynucleotides) comprise N6-methyl-adenosine (m.sup.6A). In some
embodiments, polynucleotides (e.g., RNA polynucleotides, such as
mRNA polynucleotides) comprise N6-methyl-adenosine (m.sup.6A) and
5-methyl-cytidine (m.sup.5C).
[0168] In some embodiments, polynucleotides (e.g., RNA
polynucleotides, such as mRNA polynucleotides) are uniformly
modified (e.g., fully modified, modified throughout the entire
sequence) for a particular modification. For example, a
polynucleotide can be uniformly modified with 5-methyl-cytidine
(m.sup.5C), meaning that all cytosine residues in the mRNA sequence
are replaced with 5-methyl-cytidine (m.sup.5C). Similarly, a
polynucleotide can be uniformly modified for any type of nucleoside
residue present in the sequence by replacement with a modified
residue such as those set forth above.
[0169] Exemplary nucleobases and nucleosides having a modified
cytosine include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine
(m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine),
5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine,
2-thio-cytidine (s2C), and 2-thio-5-methyl-cytidine.
[0170] In some embodiments, a modified nucleobase is a modified
uridine. In some embodiments, a modified nucleobase is a modified
cytosine. nucleosides having a modified uridine include 5-cyano
uridine and 4'-thio uridine.
[0171] In some embodiments, a modified nucleobase is a modified
adenine. Exemplary nucleobases and nucleosides having a modified
adenine include 7-deaza-adenine, 1-methyl-adenosine (m1A),
2-methyl-adenine (m2A), and N6-methyl-adenosine (m6A).
[0172] In some embodiments, a modified nucleobase is a modified
guanine. Exemplary nucleobases and nucleosides having a modified
guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG),
methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine
(preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1),
7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G),
8-oxo-guanosine, 7-methyl-8-oxo-guanosine.
[0173] The polynucleotides of the present disclosure may be
partially or fully modified along the entire length of the
molecule. For example, one or more or all or a given type of
nucleotide (e.g., purine or pyrimidine, or any one or more or all
of A, G, U, C) may be uniformly modified in a polynucleotide of the
disclosure, or in a given predetermined sequence region thereof
(e.g., in the mRNA including or excluding the polyA tail). In some
embodiments, all nucleotides X in a polynucleotide of the present
disclosure (or in a given sequence region thereof) are modified
nucleotides, wherein X may any one of nucleotides A, G, U, C, or
any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U,
A+G+C, G+U+C or A+G+C.
[0174] The polynucleotide may contain from about 1% to about 100%
modified nucleotides (either in relation to overall nucleotide
content, or in relation to one or more types of nucleotide, i.e.,
any one or more of A, G, U or C) or any intervening percentage
(e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to
60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to
95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to
60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to
95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20%
to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20%
to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from
50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%,
from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to
100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90%
to 95%, from 90% to 100%, and from 95% to 100%). Any remaining
percentage is accounted for by the presence of unmodified A, G, U,
or C.
[0175] The polynucleotides may contain at a minimum 1% and at
maximum 100% modified nucleotides, or any intervening percentage,
such as at least 5% modified nucleotides, at least 10% modified
nucleotides, at least 25% modified nucleotides, at least 50%
modified nucleotides, at least 80% modified nucleotides, or at
least 90% modified nucleotides. For example, the polynucleotides
may contain a modified pyrimidine such as a modified uracil or
cytosine. In some embodiments, at least 5%, at least 10%, at least
25%, at least 50%, at least 80%, at least 90% or 100% of the uracil
in the polynucleotide is replaced with a modified uracil (e.g., a
5-substituted uracil). The modified uracil can be replaced by a
compound having a single unique structure, or can be replaced by a
plurality of compounds having different structures (e.g., 2, 3, 4
or more unique structures). n some embodiments, at least 5%, at
least 10%, at least 25%, at least 50%, at least 80%, at least 90%
or 100% of the cytosine in the polynucleotide is replaced with a
modified cytosine (e.g., a 5-substituted cytosine). The modified
cytosine can be replaced by a compound having a single unique
structure, or can be replaced by a plurality of compounds having
different structures (e.g., 2, 3, 4 or more unique structures).
[0176] Thus, in some embodiments, the RNA (e.g., mRNA) vaccines
comprise a 5'UTR element, an optionally codon optimized open
reading frame, and a 3'UTR element, a poly(A) sequence and/or a
polyadenylation signal wherein the RNA is not chemically
modified.
[0177] In some embodiments, the modified nucleobase is a modified
uracil. Exemplary nucleobases and nucleosides having a modified
uracil include pseudouridine (.psi.), pyridin-4-one ribonucleoside,
5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine
(s.sup.2U), 4-thio-uridine (s.sup.4U), 4-thio-pseudouridine,
2-thio-pseudouridine, 5-hydroxy-uridine (ho.sup.5U),
5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor
5-bromo-uridine), 3-methyl-uridine (m.sup.3U), 5-methoxy-uridine
(mo.sup.5U), uridine 5-oxyacetic acid (cmo.sup.5U), uridine
5-oxyacetic acid methyl ester (mcmo.sup.5U),
5-carboxymethyl-uridine (cm.sup.5U), 1-carboxymethyl-pseudouridine,
5-carboxyhydroxymethyl-uridine (chm.sup.5U),
5-carboxyhydroxymethyl-uridine methyl ester (mchm.sup.5U),
5-methoxycarbonylmethyl-uridine (mcm.sup.5U),
5-methoxycarbonylmethyl-2-thio-uridine (mcm.sup.5s.sup.2U),
5-aminomethyl-2-thio-uridine (nm.sup.5s.sup.2U),
5-methylaminomethyl-uridine (mnm.sup.5U),
5-methylaminomethyl-2-thio-uridine (mnm.sup.5s.sup.2U),
5-methylaminomethyl-2-seleno-uridine (mnm.sup.5se.sup.2U),
5-carbamoylmethyl-uridine (ncm.sup.5U),
5-carboxymethylaminomethyl-uridine (cmnm.sup.5U),
5-carboxymethylaminomethyl-2-thio-uridine (cmnm.sup.5s.sup.2U),
5-propynyl-uridine, 1-propynyl-pseudouridine,
5-taurinomethyl-uridine (.tau.m.sup.5U),
1-taurinomethyl-pseudouridine,
5-taurinomethyl-2-thio-uridine(.tau.m.sup.5s.sup.2U),
1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m.sup.5U,
i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine
(m.sup.1.psi.), 5-methyl-2-thio-uridine (m.sup.5s.sup.2U),
1-methyl-4-thio-pseudouridine (m.sup.1s.sup.4.psi.),
4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine
(m.sup.3.psi.), 2-thio-1-methyl-pseudouridine,
1-methyl-1-deaza-pseudouridine,
2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),
dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine
(m.sup.5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine,
2-methoxy-uridine, 2-methoxy-4-thio-uridine,
4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine,
N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine
(acp.sup.3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine
(acp.sup.3.psi.), 5-(isopentenylaminomethyl)uridine (inm.sup.5U),
5-(isopentenylaminomethyl)-2-thio-uridine (inm.sup.5s.sup.2U),
.alpha.-thio-uridine, 2'-O-methyl-uridine (Um),
5,2'-O-dimethyl-uridine (m.sup.5Um), 2'-O-methyl-pseudouridine
(.psi.m), 2-thio-2'-O-methyl-uridine (s.sup.2Um),
5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm.sup.5Um),
5-carbamoylmethyl-2'-O-methyl-uridine (ncm.sup.5Um),
5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm.sup.5Um),
3,2'-O-dimethyl-uridine (m.sup.3Um), and
5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm.sup.5Um),
1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine,
2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and
5-[3-(1-E-propenylamino)]uridine.
[0178] In some embodiments, the modified nucleobase is a modified
cytosine. Exemplary nucleobases and nucleosides having a modified
cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine,
3-methyl-cytidine (m.sup.3C), N4-acetyl-cytidine (ac.sup.4C),
5-formyl-cytidine (f.sup.5C), N4-methyl-cytidine (m.sup.4C),
5-methyl-cytidine (m.sup.5C), 5-halo-cytidine (e.g.,
5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm.sup.5C),
1-methyl-pseudoisocytidine, pyrrolo-cytidine,
pyrrolo-pseudoisocytidine, 2-thio-cytidine (s.sup.2C),
2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,
4-thio-1-methyl-pseudoisocytidine,
4-thio-1-methyl-1-deaza-pseudoisocytidine,
1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,
5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,
2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,
4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,
lysidine (k.sub.2C), .alpha.-thio-cytidine, 2'-O-methyl-cytidine
(Cm), 5,2'-O-dimethyl-cytidine (m.sup.5Cm),
N4-acetyl-2'-O-methyl-cytidine (ac.sup.4Cm),
N4,2'-O-dimethyl-cytidine (m.sup.4Cm),
5-formyl-2'-O-methyl-cytidine (f.sup.5Cm),
N4,N4,2'-O-trimethyl-cytidine (m.sup.4.sub.2Cm), 1-thio-cytidine,
2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.
[0179] In some embodiments, the modified nucleobase is a modified
adenine. Exemplary nucleobases and nucleosides having a modified
adenine include 2-amino-purine, 2, 6-diaminopurine,
2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine),
6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine,
8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine,
7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine,
7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,
1-methyl-adenosine (m.sup.1A), 2-methyl-adenine (m.sup.2A),
N6-methyl-adenosine (m.sup.6A), 2-methylthio-N6-methyl-adenosine
(ms.sup.2 m.sup.6A), N6-isopentenyl-adenosine (i.sup.6A),
2-methylthio-N6-isopentenyl-adenosine (ms.sup.2i.sup.6A),
N6-(cis-hydroxyisopentenyl)adenosine (io.sup.6A),
2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine
(ms.sup.2io.sup.6A), N6-glycinylcarbamoyl-adenosine (g.sup.6A),
N6-threonylcarbamoyl-adenosine (t.sup.6A),
N6-methyl-N6-threonylcarbamoyl-adenosine (m.sup.6t.sup.6A)
2-methylthio-N6-threonylcarbamoyl-adenosine (ms.sup.2g.sup.6A),
N6,N6-dimethyl-adenosine (m.sup.6.sub.2A),
N6-hydroxynorvalylcarbamoyl-adenosine (hn.sup.6A),
2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine
(ms.sup.2hn.sup.6A), N6-acetyl-adenosine (ac.sup.6A),
7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine,
.alpha.-thio-adenosine, 2'-O-methyl-adenosine (Am),
N6,2'-O-dimethyl-adenosine (m.sup.6Am),
N6,N6,2'-O-trimethyl-adenosine (m.sup.6.sub.2Am),
1,2'-O-dimethyl-adenosine (m.sup.1Am), 2'-O-ribosyladenosine
(phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine,
8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine,
2'-OH-ara-adenosine, and
N6-(19-amino-pentaoxanonadecyl)-adenosine.
[0180] In some embodiments, the modified nucleobase is a modified
guanine. Exemplary nucleobases and nucleosides having a modified
guanine include inosine (I), 1-methyl-inosine (m.sup.1I), wyosine
(imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14),
isowyosine (imG2), wybutosine (yW), peroxywybutosine (o.sub.2yW),
hydroxywybutosine (OhyW), undermodified hydroxywybutosine (OhyW*),
7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ),
galactosyl-queuosine (galQ), mannosyl-queuosine (manQ),
7-cyano-7-deaza-guanosine (preQ.sub.0),
7-aminomethyl-7-deaza-guanosine (preQ.sub.1), archaeosine
(G.sup.+), 7-deaza-8-aza-guanosine, 6-thio-guanosine,
6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine,
7-methyl-guanosine (m.sup.7G), 6-thio-7-methyl-guanosine,
7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine
(m.sup.1G), N2-methyl-guanosine (m.sup.2G),
N2,N2-dimethyl-guanosine (m.sup.2.sub.2G), N2,7-dimethyl-guano sine
(m.sup.2,7G), N2, N2,7-dimethyl-guanosine (m.sup.2,2,7G),
8-oxo-guanosine, 7-methyl-8-oxo-guanosine,
1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine,
N2,N2-dimethyl-6-thio-guanosine, .alpha.-thio-guanosine,
2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine
(m.sup.2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine
(m.sup.2.sub.2Gm), 1-methyl-2'-O-methyl-guanosine (m.sup.1Gm),
N2,7-dimethyl-2'-O-methyl-guanosine (m.sup.2,7Gm),
2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m.sup.1Im),
2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine,
O6-methyl-guanosine, 2'-F-ara-guanosine, and 2'-F-guanosine.
Untranslated Regions (UTRs)
[0181] The nucleic acids of the present disclosure may comprise one
or more regions or parts which act or function as an untranslated
region. Where nucleic acids are designed to encode at least one
antigen of interest, the nucleic may comprise one or more of these
untranslated regions (UTRs). Wild-type untranslated regions of a
nucleic acid are transcribed but not translated. In mRNA, the 5'
UTR starts at the transcription start site and continues to the
start codon but does not include the start codon; whereas, the 3'
UTR starts immediately following the stop codon and continues until
the transcriptional termination signal. There is growing body of
evidence about the regulatory roles played by the UTRs in terms of
stability of the nucleic acid molecule and translation. The
regulatory features of a UTR can be incorporated into the
polynucleotides of the present disclosure to, among other things,
enhance the stability of the molecule. The specific features can
also be incorporated to ensure controlled down-regulation of the
transcript in case they are misdirected to undesired organs sites.
A variety of 5'UTR and 3'UTR sequences are known and available in
the art.
[0182] A 5' UTR is region of an mRNA that is directly upstream (5')
from the start codon (the first codon of an mRNA transcript
translated by a ribosome). A 5' UTR does not encode a protein (is
non-coding). Natural 5'UTRs have features that play roles in
translation initiation. They harbor signatures like Kozak sequences
which are commonly known to be involved in the process by which the
ribosome initiates translation of many genes. Kozak sequences have
the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or
guanine) three bases upstream of the start codon (AUG), which is
followed by another `G`. 5'UTR also have been known to form
secondary structures which are involved in elongation factor
binding.
[0183] In some embodiments of the disclosure, a 5' UTR is a
heterologous UTR, i.e., is a UTR found in nature associated with a
different ORF. In another embodiment, a 5' UTR is a synthetic UTR,
i.e., does not occur in nature. Synthetic UTRs include UTRs that
have been mutated to improve their properties, e.g., which increase
gene expression as well as those which are completely synthetic.
Exemplary 5' UTRs include Xenopus or human derived a-globin or
b-globin (U.S. Pat. Nos. 8,278,063; 9,012,219), human cytochrome
b-245 a polypeptide, and hydroxysteroid (17b) dehydrogenase, and
Tobacco etch virus (U.S. Pat. Nos. 8,278,063, 9,012,219). CMV
immediate-early 1 (IE1) gene (US2014/0206753, WO2013/185069), the
sequence GGGAUCCUACC (SEQ ID NO: 68) (WO2014/144196) may also be
used. In another embodiment, 5' UTR of a TOP gene is a 5' UTR of a
TOP gene lacking the 5' TOP motif (the oligopyrimidine tract)
(e.g., WO2015/101414, WO2015/101415, WO2015/062738, WO2015/024667,
WO2015/024668); 5' UTR element derived from ribosomal protein Large
32 (L32) gene (WO2015/101414, WO2015/101415, WO2015/062738), 5' UTR
element derived from the 5'UTR of an hydroxysteroid (1743)
dehydrogenase 4 gene (HSD17B4) (WO2015/024667), or a 5' UTR element
derived from the 5' UTR of ATP5A1 (WO2015/024667) can be used. In
some embodiments, an internal ribosome entry site (IRES) is used
instead of a 5' UTR.
[0184] In some embodiments, a 5' UTR of the present disclosure
comprises one of SEQ ID NO: 54 or SEQ ID NO: 69.
[0185] A 3' UTR is region of an mRNA that is directly downstream
(3') from the stop codon (the codon of an mRNA transcript that
signals a termination of translation). A 3' UTR does not encode a
protein (is non-coding). Natural or wild type 3' UTRs are known to
have stretches of adenosines and uridines embedded in them. These
AU rich signatures are particularly prevalent in genes with high
rates of turnover. Based on their sequence features and functional
properties, the AU rich elements (AREs) can be separated into three
classes (Chen et al, 1995): Class I AREs contain several dispersed
copies of an AUUUA motif within U-rich regions. C-Myc and MyoD
contain class I AREs. Class II AREs possess two or more overlapping
UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs
include GM-CSF and TNF-.alpha.. Class III ARES are less well
defined. These U rich regions do not contain an AUUUA motif. c-Jun
and Myogenin are two well-studied examples of this class. Most
proteins binding to the AREs are known to destabilize the
messenger, whereas members of the ELAV family, most notably HuR,
have been documented to increase the stability of mRNA. HuR binds
to AREs of all the three classes. Engineering the HuR specific
binding sites into the 3' UTR of nucleic acid molecules will lead
to HuR binding and thus, stabilization of the message in vivo.
[0186] Introduction, removal or modification of 3' UTR AU rich
elements (AREs) can be used to modulate the stability of nucleic
acids (e.g., RNA) of the disclosure. When engineering specific
nucleic acids, one or more copies of an ARE can be introduced to
make nucleic acids of the disclosure less stable and thereby
curtail translation and decrease production of the resultant
protein. Likewise, AREs can be identified and removed or mutated to
increase the intracellular stability and thus increase translation
and production of the resultant protein. Transfection experiments
can be conducted in relevant cell lines, using nucleic acids of the
disclosure and protein production can be assayed at various time
points post-transfection. For example, cells can be transfected
with different ARE-engineering molecules and by using an ELISA kit
to the relevant protein and assaying protein produced at 6 hour, 12
hour, 24 hour, 48 hour, and 7 days post-transfection. 3' UTRs may
be heterologous or synthetic. With respect to 3' UTRs, globin UTRs,
including Xenopus .beta.-globin UTRs and human .beta.-globin UTRs
are known in the art (U.S. Pat. Nos. 8,278,063, 9,012,219,
US2011/0086907). A modified .beta.-globin construct with enhanced
stability in some cell types by cloning two sequential human
.beta.-globin 3'UTRs head to tail has been developed and is well
known in the art (US2012/0195936, WO2014/071963). In addition
a2-globin, a1-globin, UTRs and mutants thereof are also known in
the art (WO2015/101415, WO2015/024667). Other 3' UTRs described in
the mRNA constructs in the non-patent literature include CYBA
(Ferizi et al., 2015) and albumin (Thess et al., 2015). Other
exemplary 3' UTRs include that of bovine or human growth hormone
(wild type or modified) (WO2013/185069, US2014/0206753,
WO2014/152774), rabbit .beta. globin and hepatitis B virus (HBV),
.alpha.-globin 3' UTR and Viral VEEV 3' UTR sequences are also
known in the art. In some embodiments, the sequence UUUGAAUU
(WO2014/144196) is used. In some embodiments, 3' UTRs of human and
mouse ribosomal protein are used. Other examples include rps9 3'UTR
(WO2015/101414), FIG. 4 (WO2015/101415), and human albumin 7
(WO2015/101415).
[0187] In some embodiments, a 3' UTR of the present disclosure
comprises SEQ ID NO: 55.
[0188] Those of ordinary skill in the art will understand that
5'UTRs that are heterologous or synthetic may be used with any
desired 3' UTR sequence. For example, a heterologous 5'UTR may be
used with a synthetic 3'UTR with a heterologous 3'' UTR.
[0189] Non-UTR sequences may also be used as regions or subregions
within a nucleic acid. For example, introns or portions of introns
sequences may be incorporated into regions of nucleic acid of the
disclosure. Incorporation of intronic sequences may increase
protein production as well as nucleic acid levels.
[0190] Combinations of features may be included in flanking regions
and may be contained within other features. For example, the ORF
may be flanked by a 5' UTR which may contain a strong Kozak
translational initiation signal and/or a 3' UTR which may include
an oligo(dT) sequence for templated addition of a poly-A tail. 5'
UTR may comprise a first polynucleotide fragment and a second
polynucleotide fragment from the same and/or different genes such
as the 5' UTRs described in US Patent Application Publication No.
2010/0293625 and PCT/US2014/069155, herein incorporated by
reference in its entirety.
[0191] It should be understood that any UTR from any gene may be
incorporated into the regions of a nucleic acid. Furthermore,
multiple wild-type UTRs of any known gene may be utilized. It is
also within the scope of the present disclosure to provide
artificial UTRs which are not variants of wild type regions. These
UTRs or portions thereof may be placed in the same orientation as
in the transcript from which they were selected or may be altered
in orientation or location. Hence a 5' or 3' UTR may be inverted,
shortened, lengthened, made with one or more other 5' UTRs or 3'
UTRs. As used herein, the term "altered" as it relates to a UTR
sequence, means that the UTR has been changed in some way in
relation to a reference sequence. For example, a 3' UTR or 5' UTR
may be altered relative to a wild-type or native UTR by the change
in orientation or location as taught above or may be altered by the
inclusion of additional nucleotides, deletion of nucleotides,
swapping or transposition of nucleotides. Any of these changes
producing an "altered" UTR (whether 3' or 5') comprise a variant
UTR.
[0192] In some embodiments, a double, triple or quadruple UTR such
as a 5' UTR or 3' UTR may be used. As used herein, a "double" UTR
is one in which two copies of the same UTR are encoded either in
series or substantially in series. For example, a double
beta-globin 3' UTR may be used as described in US Patent
publication 2010/0129877, the contents of which are incorporated
herein by reference in its entirety.
[0193] It is also within the scope of the present disclosure to
have patterned UTRs. As used herein "patterned UTRs" are those UTRs
which reflect a repeating or alternating pattern, such as ABABAB or
AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice,
or more than 3 times. In these patterns, each letter, A, B, or C
represent a different UTR at the nucleotide level.
[0194] In some embodiments, flanking regions are selected from a
family of transcripts whose proteins share a common function,
structure, feature or property. For example, polypeptides of
interest may belong to a family of proteins which are expressed in
a particular cell, tissue or at some time during development. The
UTRs from any of these genes may be swapped for any other UTR of
the same or different family of proteins to create a new
polynucleotide. As used herein, a "family of proteins" is used in
the broadest sense to refer to a group of two or more polypeptides
of interest which share at least one function, structure, feature,
localization, origin, or expression pattern.
The untranslated region may also include translation enhancer
elements (TEE). As a non-limiting example, the TEE may include
those described in US Application No. 2009/0226470, herein
incorporated by reference in its entirety, and those known in the
art. In Vitro Transcription of RNA (e.g., mRNA)
[0195] cDNA encoding the polynucleotides described herein may be
transcribed using an in vitro transcription (IVT) system. In vitro
transcription of RNA is known in the art and is described in
International Publication WO2014/152027, which is incorporated by
reference herein in its entirety.
[0196] In some embodiments, the RNA transcript is generated using a
non-amplified, linearized DNA template in an in vitro transcription
reaction to generate the RNA transcript. In some embodiments, the
template DNA is isolated DNA. In some embodiments, the template DNA
is cDNA. In some embodiments, the cDNA is formed by reverse
transcription of a RNA polynucleotide, for example, but not limited
to RNA encoding an antigenic polypeptide, e.g. mRNA. In some
embodiments, cells, e.g., bacterial cells, e.g., E. coli, e.g.,
DH-1 cells are transfected with the plasmid DNA template. In some
embodiments, the transfected cells are cultured to replicate the
plasmid DNA which is then isolated and purified. In some
embodiments, the DNA template includes a RNA polymerase promoter,
e.g., a T7 promoter located 5 ` to and operably linked to the gene
of interest.
[0197] In some embodiments, an in vitro transcription template
encodes a 5` untranslated (UTR) region, contains an open reading
frame, and encodes a 3' UTR and a polyA tail. The particular
nucleic acid sequence composition and length of an in vitro
transcription template will depend on the mRNA encoded by the
template.
[0198] A "5' untranslated region" (5'UTR) refers to a region of an
mRNA that is directly upstream (i.e., 5') from the start codon
(i.e., the first codon of an mRNA transcript translated by a
ribosome) that does not encode a polypeptide. When RNA transcripts
are being generated, the 5' UTR may comprise a promoter sequence.
Such promoter sequences are known in the art. It should be
understood that such promoter sequences will not be present in a
vaccine of the disclosure.
[0199] A "3' untranslated region" (3'UTR) refers to a region of an
mRNA that is directly downstream (i.e., 3') from the stop codon
(i.e., the codon of an mRNA transcript that signals a termination
of translation) that does not encode a polypeptide.
[0200] An "open reading frame" is a continuous stretch of DNA
beginning with a start codon (e.g., methionine (ATG)), and ending
with a stop codon (e.g., TAA, TAG or TGA) and encodes a
polypeptide.
[0201] A "polyA tail" is a region of mRNA that is downstream, e.g.,
directly downstream (i.e., 3'), from the 3' UTR that contains
multiple, consecutive adenosine monophosphates. A polyA tail may
contain 10 to 300 adenosine monophosphates. For example, a polyA
tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,
130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,
260, 270, 280, 290 or 300 adenosine monophosphates. In some
embodiments, a polyA tail contains 50 to 250 adenosine
monophosphates. In a relevant biological setting (e.g., in cells,
in vivo) the poly(A) tail functions to protect mRNA from enzymatic
degradation, e.g., in the cytoplasm, and aids in transcription
termination, export of the mRNA from the nucleus and
translation.
[0202] In some embodiments, a polynucleotide includes 200 to 3,000
nucleotides. For example, a polynucleotide may include 200 to 500,
200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500,
500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000,
1500 to 3000, or 2000 to 3000 nucleotides.
[0203] An in vitro transcription system typically comprises a
transcription buffer, nucleotide triphosphates (NTPs), an RNase
inhibitor and a polymerase.
[0204] The NTPs may be manufactured in house, may be selected from
a supplier, or may be synthesized as described herein. The NTPs may
be selected from, but are not limited to, those described herein
including natural and unnatural (modified) NTPs.
[0205] Any number of RNA polymerases or variants may be used in the
method of the present disclosure. The polymerase may be selected
from, but is not limited to, a phage RNA polymerase, e.g., a T7 RNA
polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, and/or
mutant polymerases such as, but not limited to, polymerases able to
incorporate modified nucleic acids and/or modified nucleotides,
including chemically modified nucleic acids and/or nucleotides.
Some embodiments exclude the use of DNase.
[0206] In some embodiments, the RNA transcript is capped via
enzymatic capping. In some embodiments, the RNA comprises 5'
terminal cap, for example, 7mG(5')ppp(5')NlmpNp.
Chemical Synthesis
[0207] Solid-Phase Chemical Synthesis.
[0208] Nucleic acids the present disclosure may be manufactured in
whole or in part using solid phase techniques. Solid-phase chemical
synthesis of nucleic acids is an automated method wherein molecules
are immobilized on a solid support and synthesized step by step in
a reactant solution. Solid-phase synthesis is useful in
site-specific introduction of chemical modifications in the nucleic
acid sequences.
[0209] Liquid Phase Chemical Synthesis.
[0210] The synthesis of nucleic acids of the present disclosure by
the sequential addition of monomer building blocks may be carried
out in a liquid phase.
[0211] Combination of Synthetic Methods.
[0212] The synthetic methods discussed above each has its own
advantages and limitations. Attempts have been conducted to combine
these methods to overcome the limitations. Such combinations of
methods are within the scope of the present disclosure. The use of
solid-phase or liquid-phase chemical synthesis in combination with
enzymatic ligation provides an efficient way to generate long chain
nucleic acids that cannot be obtained by chemical synthesis
alone.
Ligation of Nucleic Acid Regions or Subregions
[0213] Assembling nucleic acids by a ligase may also be used. DNA
or RNA ligases promote intermolecular ligation of the 5' and 3'
ends of polynucleotide chains through the formation of a
phosphodiester bond. Nucleic acids such as chimeric polynucleotides
and/or circular nucleic acids may be prepared by ligation of one or
more regions or subregions. DNA fragments can be joined by a ligase
catalyzed reaction to create recombinant DNA with different
functions. Two oligodeoxynucleotides, one with a 5' phosphoryl
group and another with a free 3' hydroxyl group, serve as
substrates for a DNA ligase.
Purification
[0214] Purification of the nucleic acids described herein may
include, but is not limited to, nucleic acid clean-up, quality
assurance and quality control. Clean-up may be performed by methods
known in the arts such as, but not limited to, AGENCOURT.RTM. beads
(Beckman Coulter Genomics, Danvers, Mass.), poly-T beads, LNA.TM.
oligo-T capture probes (EXIQON.RTM. Inc, Vedbaek, Denmark) or HPLC
based purification methods such as, but not limited to, strong
anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC
(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term
"purified" when used in relation to a nucleic acid such as a
"purified nucleic acid" refers to one that is separated from at
least one contaminant. A "contaminant" is any substance that makes
another unfit, impure or inferior. Thus, a purified nucleic acid
(e.g., DNA and RNA) is present in a form or setting different from
that in which it is found in nature, or a form or setting different
from that which existed prior to subjecting it to a treatment or
purification method.
[0215] A quality assurance and/or quality control check may be
conducted using methods such as, but not limited to, gel
electrophoresis, UV absorbance, or analytical HPLC.
[0216] In some embodiments, the nucleic acids may be sequenced by
methods including, but not limited to
reverse-transcriptase-PCR.
Quantification
[0217] In some embodiments, the nucleic acids of the present
invention may be quantified in exosomes or when derived from one or
more bodily fluid. Bodily fluids include peripheral blood, serum,
plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva,
bone marrow, synovial fluid, aqueous humor, amniotic fluid,
cerumen, breast milk, broncheoalveolar lavage fluid, semen,
prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat,
fecal matter, hair, tears, cyst fluid, pleural and peritoneal
fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial
fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal
secretion, stool water, pancreatic juice, lavage fluids from sinus
cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and
umbilical cord blood. Alternatively, exosomes may be retrieved from
an organ selected from the group consisting of lung, heart,
pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin,
colon, breast, prostate, brain, esophagus, liver, and placenta.
[0218] Assays may be performed using construct specific probes,
cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry,
electrophoresis, mass spectrometry, or combinations thereof while
the exosomes may be isolated using immunohistochemical methods such
as enzyme linked immunosorbent assay (ELISA) methods. Exosomes may
also be isolated by size exclusion chromatography, density gradient
centrifugation, differential centrifugation, nanomembrane
ultrafiltration, immunoabsorbent capture, affinity purification,
microfluidic separation, or combinations thereof.
[0219] These methods afford the investigator the ability to
monitor, in real time, the level of nucleic acids remaining or
delivered. This is possible because the nucleic acids of the
present disclosure, in some embodiments, differ from the endogenous
forms due to the structural or chemical modifications.
[0220] In some embodiments, the nucleic acid may be quantified
using methods such as, but not limited to, ultraviolet visible
spectroscopy (UV/Vis). A non-limiting example of a UV/Vis
spectrometer is a NANODROP.RTM. spectrometer (ThermoFisher,
Waltham, Mass.). The quantified nucleic acid may be analyzed in
order to determine if the nucleic acid may be of proper size, check
that no degradation of the nucleic acid has occurred. Degradation
of the nucleic acid may be checked by methods such as, but not
limited to, agarose gel electrophoresis, HPLC based purification
methods such as, but not limited to, strong anion exchange HPLC,
weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and
hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-mass
spectrometry (LCMS), capillary electrophoresis (CE) and capillary
gel electrophoresis (CGE).
Flagellin Adjuvants
[0221] Flagellin is an approximately 500 amino acid monomeric
protein that polymerizes to form the flagella associated with
bacterial motion. Flagellin is expressed by a variety of
flagellated bacteria (Salmonella typhimurium for example) as well
as non-flagellated bacteria (such as Escherichia coli). Sensing of
flagellin by cells of the innate immune system (dendritic cells,
macrophages, etc.) is mediated by the Toll-like receptor 5 (TLR5)
as well as by Nod-like receptors (NLRs) Ipaf and Naip5. TLRs and
NLRs have been identified as playing a role in the activation of
innate immune response and adaptive immune response. As such,
flagellin provides an adjuvant effect in a vaccine.
[0222] The nucleotide and amino acid sequences encoding known
flagellin polypeptides are publicly available in the NCBI GenBank
database. The flagellin sequences from S. Typhimurium, H. Pylori,
V. Cholera, S. marcesens, S. flexneri, T. Pallidum, L. pneumophila,
B. burgdorferei, C. difficile, R. meliloti, A. tumefaciens, R.
lupini, B. clarridgeiae, P. Mirabilis, B. subtilus, L.
monocytogenes, P. aeruginosa, and E. coli, among others are
known.
[0223] A flagellin polypeptide, as used herein, refers to a full
length flagellin protein, immunogenic fragments thereof, and
peptides having at least 50% sequence identify to a flagellin
protein or immunogenic fragments thereof. Exemplary flagellin
proteins include flagellin from Salmonella typhi (UniPro Entry
number: Q56086), Salmonella typhimurium (A0A0C9DG09), Salmonella
enteritidis (A0A0C9BAB7), and Salmonella choleraesuis (Q6V2X8), and
SEQ ID NO: 47-49. In some embodiments, the flagellin polypeptide
has at least 60%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, or 99%
sequence identify to a flagellin protein or immunogenic fragments
thereof.
[0224] In some embodiments, the flagellin polypeptide is an
immunogenic fragment. An immunogenic fragment is a portion of a
flagellin protein that provokes an immune response. In some
embodiments, the immune response is a TLR5 immune response. An
example of an immunogenic fragment is a flagellin protein in which
all or a portion of a hinge region has been deleted or replaced
with other amino acids. For example, an antigenic polypeptide may
be inserted in the hinge region. Hinge regions are the
hypervariable regions of a flagellin. Hinge regions of a flagellin
are also referred to as "D3 domain or region, "propeller domain or
region," "hypervariable domain or region" and "variable domain or
region." "At least a portion of a hinge region," as used herein,
refers to any part of the hinge region of the flagellin, or the
entirety of the hinge region. In other embodiments an immunogenic
fragment of flagellin is a 20, 25, 30, 35, or 40 amino acid
C-terminal fragment of flagellin.
[0225] The flagellin monomer is formed by domains D0 through D3. D0
and D1, which form the stem, are composed of tandem long alpha
helices and are highly conserved among different bacteria. The D1
domain includes several stretches of amino acids that are useful
for TLR5 activation. The entire D1 domain or one or more of the
active regions within the domain are immunogenic fragments of
flagellin. Examples of immunogenic regions within the D1 domain
include residues 88-114 and residues 411-431 (in Salmonella
typhimurium FliC flagellin). Within the 13 amino acids in the
88-100 region, at least 6 substitutions are permitted between
Salmonella flagellin and other flagellins that still preserve TLR5
activation. Thus, immunogenic fragments of flagellin include
flagellin like sequences that activate TLR5 and contain a 13 amino
acid motif that is 53% or more identical to the Salmonella sequence
in 88-100 of FliC (LQRVRELAVQSAN; SEQ ID NO: 53).
[0226] In some embodiments, the RNA (e.g., mRNA) vaccine includes
an RNA that encodes a fusion protein of flagellin and one or more
antigenic polypeptides. A "fusion protein" as used herein, refers
to a linking of two components of the construct. In some
embodiments, a carboxy-terminus of the antigenic polypeptide is
fused or linked to an amino terminus of the flagellin polypeptide.
In other embodiments, an amino-terminus of the antigenic
polypeptide is fused or linked to a carboxy-terminus of the
flagellin polypeptide. The fusion protein may include, for example,
one, two, three, four, five, six or more flagellin polypeptides
linked to one, two, three, four, five, six or more antigenic
polypeptides. When two or more flagellin polypeptides and/or two or
more antigenic polypeptides are linked such a construct may be
referred to as a "multimer."
[0227] Each of the components of a fusion protein may be directly
linked to one another or they may be connected through a linker.
For instance, the linker may be an amino acid linker. The amino
acid linker encoded for by the RNA (e.g., mRNA) vaccine to link the
components of the fusion protein may include, for instance, at
least one member selected from the group consisting of a lysine
residue, a glutamic acid residue, a serine residue and an arginine
residue. In some embodiments the linker is 1-30, 1-25, 1-25, 5-10,
5, 15, or 5-20 amino acids in length.
[0228] In other embodiments the RNA (e.g., mRNA) vaccine includes
at least two separate RNA polynucleotides, one encoding one or more
antigenic polypeptides and the other encoding the flagellin
polypeptide. The at least two RNA polynucleotides may be
co-formulated in a carrier such as a lipid nanoparticle.
Broad Spectrum RNA (e.g., mRNA) Vaccines
[0229] There may be situations where persons are at risk for
infection with more than one strain of Streptococcus,
Staphylococcus, or other bacteria. RNA (e.g., mRNA) therapeutic
vaccines are particularly amenable to combination vaccination
approaches due to a number of factors including, but not limited
to, speed of manufacture, ability to rapidly tailor vaccines to
accommodate perceived geographical threat, and the like. Moreover,
because the vaccines utilize the human body to produce the
antigenic protein, the vaccines are amenable to the production of
larger, more complex antigenic proteins, allowing for proper
folding, surface expression, antigen presentation, etc. in the
human subject. To protect against more than one strain of a
bacterial infection, a combination vaccine can be administered that
includes RNA (e.g., mRNA) encoding at least one antigenic
polypeptide protein (or antigenic portion thereof) of a first
bacterium and further includes RNA encoding at least one antigenic
polypeptide protein (or antigenic portion thereof) of a second
bacterium. Thus, the vaccines of the present disclosure may be
combination vaccines that target one or more antigens of the same
strain/species, or one or more antigens of different
strains/species, e.g., antigens which induce immunity to organisms
which are found in the same geographic areas where the risk of
certain bacterial infections is high or organisms to which an
individual is likely to be exposed to when exposed to a certain
bacterium. RNA (e.g., mRNA) can be co-formulated, for example, in a
single lipid nanoparticle (LNP) or can be formulated in separate
LNPs for co-administration.
Methods of Treatment
[0230] Provided herein are compositions (e.g., pharmaceutical
compositions), methods, kits and reagents for prevention and/or
treatment of bacterial infections in humans and other mammals.
Bacterial RNA (e.g. mRNA) vaccines can be used as therapeutic or
prophylactic agents, alone or in combination with other vaccine(s).
They may be used in medicine to prevent and/or treat bacterial
infections. In exemplary aspects, the RNA (e.g., mRNA) vaccines of
the present disclosure are used to provide prophylactic protection
from bacterial infections. Prophylactic protection from bacterial
infections can be achieved following administration of a RNA (e.g.,
mRNA) vaccine of the present disclosure. Bacterial RNA (e.g., mRNA)
vaccines of the present disclosure may be used to treat or prevent
"co-infections" containing two or more bacterial infections.
Vaccines can be administered once, twice, three times, four times
or more, but it is likely sufficient to administer the vaccine once
(optionally followed by a single booster). It is possible, although
less desirable, to administer the vaccine to an infected individual
to achieve a therapeutic response. Dosing may need to be adjusted
accordingly.
[0231] A method of eliciting an immune response in a subject
against one or more bacterial infections is provided in aspects of
the present disclosure. The method involves administering to the
subject a bacterial RNA (e.g., mRNA) vaccine comprising at least
one RNA (e.g., mRNA) polynucleotide having an open reading frame
encoding at least one bacterial antigenic polypeptide thereof,
thereby inducing in the subject an immune response specific to the
bacterial antigenic polypeptide or an immunogenic fragment thereof,
wherein anti-antigenic polypeptide antibody titer in the subject is
increased following vaccination relative to anti-antigenic
polypeptide antibody titer in a subject vaccinated with a
prophylactically effective dose of a traditional vaccine against
bacterial infections. An "anti-antigenic polypeptide antibody" is a
serum antibody the binds specifically to the antigenic
polypeptide.
[0232] In some embodiments, a RNA (e.g., mRNA) vaccine capable of
eliciting an immune response is administered intramuscularly via a
composition including a compound according to Formula (I), (IA),
(II), (IIa), (IIb), (IIc), (IId) or (IIe) (e.g., Compound 3, 18,
20, 25, 26, 29, 30, 60, 108-112, or 122).
[0233] A prophylactically effective dose is a therapeutically
effective dose that prevents infection with the bacteria at a
clinically acceptable level. In some embodiments the
therapeutically effective dose is a dose listed in a package insert
for the vaccine. A traditional vaccine, as used herein, refers to a
vaccine other than the RNA (e.g., mRNA) vaccines of the present
disclosure. For instance, a traditional vaccine includes but is not
limited to live/attenuated microorganism vaccines,
killed/inactivated microorganism vaccines, subunit vaccines,
protein antigen vaccines, DNA vaccines, etc. In exemplary
embodiments, a traditional vaccine is a vaccine that has achieved
regulatory approval and/or is registered by a national drug
regulatory body, for example the Food and Drug Administration (FDA)
in the United States or the European Medicines Agency (EMA).
[0234] In some embodiments the anti-antigenic polypeptide antibody
titer in the subject is increased 1 log to 10 log following
vaccination relative to anti-antigenic polypeptide antibody titer
in a subject vaccinated with a prophylactically effective dose of a
traditional vaccine against one or more bacterial infections.
[0235] In some embodiments the anti-antigenic polypeptide antibody
titer in the subject is increased 1 log, 2 log, 3 log, 5 log or 10
log following vaccination relative to anti-antigenic polypeptide
antibody titer in a subject vaccinated with a prophylactically
effective dose of a traditional vaccine against one or more
bacterial infections.
[0236] A method of eliciting an immune response in a subject
against one or more bacterial infections is provided in other
aspects of the disclosure. The method involves administering to the
subject a bacterial RNA (e.g., mRNA) vaccine comprising at least
one RNA (e.g., mRNA) polynucleotide having an open reading frame
encoding at least one bacterial antigenic polypeptide or an
immunogenic fragment thereof, thereby inducing in the subject an
immune response specific to a bacterial antigenic polypeptide or an
immunogenic fragment thereof, wherein the immune response in the
subject is equivalent to an immune response in a subject vaccinated
with a traditional vaccine against the bacterial infection at 2
times to 100 times the dosage level relative to the RNA (e.g.,
mRNA) vaccine.
[0237] In some embodiments, the immune response in the subject is
equivalent to an immune response in a subject vaccinated with a
traditional vaccine at 2, 3, 4, 5, 10, 50, 100 times the dosage
level relative to the bacterial RNA (e.g., mRNA) vaccine.
In some embodiments the immune response in the subject is
equivalent to an immune response in a subject vaccinated with a
traditional vaccine at 10-100 times, or 100-1000 times, the dosage
level relative to the bacterial RNA (e.g., mRNA) vaccine.
[0238] In some embodiments the immune response is assessed by
determining antibody titer in the subject.
[0239] Some aspects of the present disclosure provide a method of
eliciting an immune response in a subject against a In some
embodiments the immune response in the subject is equivalent to an
immune response in a subject vaccinated with a traditional vaccine
at 2, 3, 4, 5, 10, 50, 100 times the dosage level relative to the
bacterial RNA (e.g., mRNA) vaccine by administering to the subject
a bacterial RNA (e.g., mRNA) vaccine comprising at least one RNA
(e.g., mRNA) polynucleotide having an open reading frame encoding
at least one bacterial antigenic polypeptide, thereby inducing in
the subject an immune response specific to the antigenic
polypeptide or an immunogenic fragment thereof, wherein the immune
response in the subject is induced 2 days to 10 weeks earlier
relative to an immune response induced in a subject vaccinated with
a prophylactically effective dose of a traditional vaccine against
the bacterial infection(s). In some embodiments, the immune
response in the subject is induced in a subject vaccinated with a
prophylactically effective dose of a traditional vaccine at 2 times
to 100 times the dosage level relative to the RNA (e.g., mRNA)
vaccine.
[0240] In some embodiments, the immune response in the subject is
induced 2 days earlier, or 3 days earlier, relative to an immune
response induced in a subject vaccinated with a prophylactically
effective dose of a traditional vaccine.
[0241] In some embodiments the immune response in the subject is
induced 1 week, 2 weeks, 3 weeks, 5 weeks, or 10 weeks earlier
relative to an immune response induced in a subject vaccinated with
a prophylactically effective dose of a traditional vaccine.
[0242] Also provided herein is a method of eliciting an immune
response in a subject against a bacterial infection by
administering to the subject a bacterial RNA (e.g., mRNA) vaccine
having an open reading frame encoding a first antigenic
polypeptide, wherein the RNA polynucleotide does not include a
stabilization element, and wherein an adjuvant is not co-formulated
or co-administered with the vaccine.
Therapeutic and Prophylactic Compositions
[0243] Provided herein are compositions (e.g., pharmaceutical
compositions), methods, kits and reagents for prevention, treatment
or diagnosis of bacterial infections in humans and other mammals,
for example. Bacterial RNA (e.g. mRNA) vaccines can be used as
therapeutic or prophylactic agents. They may be used in medicine to
prevent and/or treat infectious disease. In some embodiments, the
bacterial RNA (e.g., mRNA) vaccines of the present disclosure are
used for the priming of immune effector cells, for example, to
activate peripheral blood mononuclear cells (PBMCs) ex vivo, which
are then infused (re-infused) into a subject.
[0244] In some embodiments, bacterial vaccine containing RNA (e.g.,
mRNA) polynucleotides as described herein can be administered to a
subject (e.g., a mammalian subject, such as a human subject), and
the RNA (e.g., mRNA) polynucleotides are translated in vivo to
produce an antigenic polypeptide.
[0245] The bacterial RNA (e.g., mRNA) vaccines may be induced for
translation of a polypeptide (e.g., antigen or immunogen) in a
cell, tissue or organism. In some embodiments, such translation
occurs in vivo, although such translation may occur ex vivo, in
culture or in vitro. In some embodiments, the cell, tissue or
organism is contacted with an effective amount of a composition
containing a bacterial RNA (e.g., mRNA) vaccine that contains a
polynucleotide that has at least one a translatable region encoding
an antigenic polypeptide.
[0246] An "effective amount" of a bacterial RNA (e.g. mRNA) vaccine
is provided based, at least in part, on the target tissue, target
cell type, means of administration, physical characteristics of the
polynucleotide (e.g., size, and extent of modified nucleosides) and
other components of the vaccine, and other determinants. In
general, an effective amount of the bacterial RNA (e.g., mRNA)
vaccine composition provides an induced or boosted immune response
as a function of antigen production in the cell, preferably more
efficient than a composition containing a corresponding unmodified
polynucleotide encoding the same antigen or a peptide antigen.
Increased antigen production may be demonstrated by increased cell
transfection (the percentage of cells transfected with the RNA,
e.g., mRNA, vaccine), increased protein translation from the
polynucleotide, decreased nucleic acid degradation (as
demonstrated, for example, by increased duration of protein
translation from a modified polynucleotide), or altered antigen
specific immune response of the host cell.
[0247] In some embodiments, RNA (e.g. mRNA) vaccines (including
polynucleotides their encoded polypeptides) in accordance with the
present disclosure may be used for treatment of bacterial
infections.
[0248] Bacterial RNA (e.g. mRNA) vaccines may be administered
prophylactically or therapeutically as part of an active
immunization scheme to healthy individuals or early in infection
during the incubation phase or during active infection after onset
of symptoms. In some embodiments, the amount of RNA (e.g., mRNA)
vaccine of the present disclosure provided to a cell, a tissue or a
subject may be an amount effective for immune prophylaxis.
[0249] Bacterial RNA (e.g. mRNA) vaccines may be administrated with
other prophylactic or therapeutic compounds. As a non-limiting
example, a prophylactic or therapeutic compound may be an adjuvant
or a booster. As used herein, when referring to a prophylactic
composition, such as a vaccine, the term "booster" refers to an
extra administration of the prophylactic (vaccine) composition. A
booster (or booster vaccine) may be given after an earlier
administration of the prophylactic composition. The time of
administration between the initial administration of the
prophylactic composition and the booster may be, but is not limited
to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6
minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes,
20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55
minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7
hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14
hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours,
21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4
days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2
months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months,
9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3
years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10
years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years,
17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35
years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years,
70 years, 75 years, 80 years, 85 years, 90 years, 95 years or more
than 99 years. In some embodiments, the time of administration
between the initial administration of the prophylactic composition
and the booster may be, but is not limited to, 1 week, 2 weeks, 3
weeks, 1 month, 2 months, 3 months, 6 months or 1 year.
[0250] In some embodiments, bacterial RNA (e.g. mRNA) vaccines may
be administered intramuscularly or intradermally, similarly to the
administration of inactivated vaccines known in the art.
[0251] Bacterial RNA (e.g. mRNA) vaccines may be utilized in
various settings depending on the prevalence of the infection or
the degree or level of unmet medical need. As a non-limiting
example, the RNA (e.g., mRNA) vaccines may be utilized to treat
and/or prevent a variety of bacterial infections. RNA (e.g., mRNA)
vaccines have superior properties in that they produce much larger
antibody titers and produce responses early than commercially
available anti-bacterial agents/compositions.
[0252] Provided herein are pharmaceutical compositions including
bacterial RNA (e.g. mRNA) vaccines and RNA (e.g. mRNA) vaccine
compositions and/or complexes optionally in combination with one or
more pharmaceutically acceptable excipients.
[0253] Bacterial RNA (e.g. mRNA) vaccines may be formulated or
administered alone or in conjunction with one or more other
components. For instance, bacterial RNA (e.g., mRNA) vaccines
(vaccine compositions) may comprise other components including, but
not limited to, adjuvants.
[0254] In some embodiments, bacterial (e.g. mRNA) vaccines do not
include an adjuvant (they are adjuvant-free).
[0255] Bacterial RNA (e.g. mRNA) vaccines may be formulated or
administered in combination with one or more
pharmaceutically-acceptable excipients. In some embodiments,
vaccine compositions comprise at least one additional active
substances, such as, for example, a therapeutically-active
substance, a prophylactically-active substance, or a combination of
both. Vaccine compositions may be sterile, pyrogen-free or both
sterile and pyrogen-free. General considerations in the formulation
and/or manufacture of pharmaceutical agents, such as vaccine
compositions, may be found, for example, in Remington: The Science
and Practice of Pharmacy 21st ed., Lippincott Williams &
Wilkins, 2005 (incorporated herein by reference in its
entirety).
[0256] In some embodiments, bacterial RNA (e.g. mRNA) vaccines are
administered to humans, human patients or subjects. For the
purposes of the present disclosure, the phrase "active ingredient"
generally refers to the RNA (e.g., mRNA) vaccines or the
polynucleotides contained therein, for example, RNA polynucleotides
(e.g., mRNA polynucleotides) encoding antigenic polypeptides.
[0257] Formulations of the bacterial vaccine compositions described
herein may be prepared by any method known or hereafter developed
in the art of pharmacology. In general, such preparatory methods
include the step of bringing the active ingredient (e.g., mRNA
polynucleotide) into association with an excipient and/or one or
more other accessory ingredients, and then, if necessary and/or
desirable, dividing, shaping and/or packaging the product into a
desired single- or multi-dose unit.
[0258] Relative amounts of the active ingredient, the
pharmaceutically acceptable excipient, and/or any additional
ingredients in a pharmaceutical composition in accordance with the
disclosure will vary, depending upon the identity, size, and/or
condition of the subject treated and further depending upon the
route by which the composition is to be administered. By way of
example, the composition may comprise between 0.1% and 100%, e.g.,
between 0.5 and 50%, between 1-30%, between 5-80%, at least 80%
(w/w) active ingredient.
[0259] Bacterial RNA (e.g. mRNA) vaccines can be formulated using
one or more excipients to: (1) increase stability; (2) increase
cell transfection; (3) permit the sustained or delayed release
(e.g., from a depot formulation); (4) alter the biodistribution
(e.g., target to specific tissues or cell types); (5) increase the
translation of encoded protein in vivo; and/or (6) alter the
release profile of encoded protein (antigen) in vivo. In addition
to traditional excipients such as any and all solvents, dispersion
media, diluents, or other liquid vehicles, dispersion or suspension
aids, surface active agents, isotonic agents, thickening or
emulsifying agents, preservatives, excipients can include, without
limitation, lipidoids, liposomes, lipid nanoparticles, polymers,
lipoplexes, core-shell nanoparticles, peptides, proteins, cells
transfected with bacterial RNA (e.g. mRNA) vaccines (e.g., for
transplantation into a subject), hyaluronidase, nanoparticle mimics
and combinations thereof.
Stabilizing Elements
[0260] Naturally-occurring eukaryotic mRNA molecules have been
found to contain stabilizing elements, including, but not limited
to untranslated regions (UTR) at their 5'-end (5'UTR) and/or at
their 3'-end (3'UTR), in addition to other structural features,
such as a 5'-cap structure or a 3'-poly(A) tail. Both the 5'UTR and
the 3'UTR are typically transcribed from the genomic DNA and are
elements of the premature mRNA. Characteristic structural features
of mature mRNA, such as the 5'-cap and the 3'-poly(A) tail are
usually added to the transcribed (premature) mRNA during mRNA
processing. The 3'-poly(A) tail is typically a stretch of adenine
nucleotides added to the 3'-end of the transcribed mRNA. It can
comprise up to about 400 adenine nucleotides. In some embodiments
the length of the 3'-poly(A) tail may be an essential element with
respect to the stability of the individual mRNA.
[0261] In some embodiments, a vaccine includes at least one RNA
polynucleotide having an open reading frame encoding at least one
antigenic polypeptide having at least one modification, at least
one 5' terminal cap, and is formulated within a lipid nanoparticle.
5'-capping of polynucleotides may be completed concomitantly during
the in vitro-transcription reaction using the following chemical
RNA cap analogs to generate the 5'-guanosine cap structure
according to manufacturer protocols: 3'-O-Me-m7G(5')ppp(5') G [the
ARCA cap]; G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A;
m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). 5'-capping
of modified RNA may be completed post-transcriptionally using a
Vaccinia Virus Capping Enzyme to generate the "Cap 0" structure:
m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). Cap 1
structure may be generated using both Vaccinia Virus Capping Enzyme
and a 2'-O methyl-transferase to generate:
m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structure may be generated from
the Cap 1 structure followed by the 2'-O-methylation of the
5'-antepenultimate nucleotide using a 2'-O methyl-transferase. Cap
3 structure may be generated from the Cap 2 structure followed by
the 2'-O-methylation of the 5'-preantepenultimate nucleotide using
a 2'-O methyl-transferase. Enzymes may be derived from a
recombinant source.
[0262] The 3'-poly(A) tail is typically a stretch of adenine
nucleotides added to the 3'-end of the transcribed mRNA. It can, in
some instances, comprise up to about 400 adenine nucleotides. In
some embodiments, the length of the 3'-poly(A) tail may be an
essential element with respect to the stability of the individual
mRNA.
[0263] In some embodiments the RNA (e.g., mRNA) vaccine may include
one or more stabilizing elements. Stabilizing elements may include
for instance a histone stem-loop. A stem-loop binding protein
(SLBP), a 32 kDa protein has been identified. It is associated with
the histone stem-loop at the 3'-end of the histone messages in both
the nucleus and the cytoplasm. Its expression level is regulated by
the cell cycle; it peaks during the S-phase, when histone mRNA
levels are also elevated. The protein has been shown to be
essential for efficient 3'-end processing of histone pre-mRNA by
the U7 snRNP. SLBP continues to be associated with the stem-loop
after processing, and then stimulates the translation of mature
histone mRNAs into histone proteins in the cytoplasm. The RNA
binding domain of SLBP is conserved through metazoa and protozoa;
its binding to the histone stem-loop depends on the structure of
the loop. The minimum binding site includes at least three
nucleotides 5' and two nucleotides 3' relative to the
stem-loop.
[0264] In some embodiments, the RNA (e.g., mRNA) vaccines include a
coding region, at least one histone stem-loop, and optionally, a
poly(A) sequence or polyadenylation signal. The poly(A) sequence or
polyadenylation signal generally should enhance the expression
level of the encoded protein. The encoded protein, in some
embodiments, is not a histone protein, a reporter protein (e.g.
Luciferase, GFP, EGFP, .beta.-Galactosidase, EGFP), or a marker or
selection protein (e.g. alpha-Globin, Galactokinase and
Xanthine:guanine phosphoribosyl transferase (GPT)).
[0265] In some embodiments, the combination of a poly(A) sequence
or polyadenylation signal and at least one histone stem-loop, even
though both represent alternative mechanisms in nature, acts
synergistically to increase the protein expression beyond the level
observed with either of the individual elements. It has been found
that the synergistic effect of the combination of poly(A) and at
least one histone stem-loop does not depend on the order of the
elements or the length of the poly(A) sequence.
[0266] In some embodiments, the RNA (e.g., mRNA) vaccine does not
comprise a histone downstream element (HDE). "Histone downstream
element" (HDE) includes a purine-rich polynucleotide stretch of
approximately 15 to 20 nucleotides 3' of naturally occurring
stem-loops, representing the binding site for the U7 snRNA, which
is involved in processing of histone pre-mRNA into mature histone
mRNA. Ideally, the inventive nucleic acid does not include an
intron.
[0267] In some embodiments, the RNA (e.g., mRNA) vaccine may or may
not contain an enhancer and/or promoter sequence, which may be
modified or unmodified or which may be activated or inactivated. In
some embodiments, the histone stem-loop is generally derived from
histone genes, and includes an intramolecular base pairing of two
neighbored partially or entirely reverse complementary sequences
separated by a spacer, including (e.g., consisting of) a short
sequence, which forms the loop of the structure. The unpaired loop
region is typically unable to base pair with either of the stem
loop elements. It occurs more often in RNA, as is a key component
of many RNA secondary structures, but may be present in
single-stranded DNA as well. Stability of the stem-loop structure
generally depends on the length, number of mismatches or bulges,
and base composition of the paired region. In some embodiments,
wobble base pairing (non-Watson-Crick base pairing) may result. In
some embodiments, the at least one histone stem-loop sequence
comprises a length of 15 to 45 nucleotides.
[0268] In other embodiments the RNA (e.g., mRNA) vaccine may have
one or more AU-rich sequences removed. These sequences, sometimes
referred to as AURES are destabilizing sequences found in the
3'UTR. The AURES may be removed from the RNA (e.g., mRNA) vaccines.
Alternatively the AURES may remain in the RNA (e.g., mRNA)
vaccine.
Nanoparticle Formulations
[0269] The lipid nanoparticle (LNP) delivery is superior to other
formulations including a protamine base approach described in the
literature and no additional adjuvants are to be necessary. The use
of LNPs enables the effective delivery of chemically modified or
unmodified mRNA vaccines. Both modified and unmodified LNP
formulated mRNA vaccines are superior to conventional vaccines by a
significant degree. In some embodiments the mRNA vaccines of the
invention are superior to conventional vaccines by a factor of at
least 10 fold, 20 fold, 40 fold, 50 fold, 100 fold, 500 fold or
1,000 fold.
[0270] The fact that LNP formulations significantly enhance the
effectiveness of mRNA vaccines, including chemically modified and
unmodified mRNA vaccines provides a baseline formulation for the
bacterial vaccines of the invention. The results presented herein
demonstrate the unexpected superior efficacy of the mRNA vaccines
formulated in LNP with mutated N-glycosylation sites.
[0271] In some embodiments, bacterial RNA (e.g. mRNA) vaccines are
formulated in a nanoparticle. In some embodiments, bacterial RNA
(e.g. mRNA) vaccines are formulated in a lipid nanoparticle. In
some embodiments, bacterial RNA (e.g. mRNA) vaccines are formulated
in a lipid-polycation complex, referred to as a cationic lipid
nanoparticle. As a non-limiting example, the polycation may include
a cationic peptide or a polypeptide such as, but not limited to,
polylysine, polyornithine and/or polyarginine. In some embodiments,
bacterial RNA (e.g., mRNA) vaccines are formulated in a lipid
nanoparticle that includes a non-cationic lipid such as, but not
limited to, cholesterol or dioleoyl phosphatidylethanolamine
(DOPE).
[0272] A lipid nanoparticle formulation may be influenced by, but
not limited to, the selection of the cationic lipid component, the
degree of cationic lipid saturation, the nature of the PEGylation,
ratio of all components and biophysical parameters such as size. In
one example by Semple et al. (Nature Biotech. 2010 28:172-176), the
lipid nanoparticle formulation is composed of 57.1% cationic lipid,
7.1% dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4%
PEG-c-DMA. As another example, changing the composition of the
cationic lipid can more effectively deliver siRNA to various
antigen presenting cells (Basha et al. Mol Ther. 2011
19:2186-2200).
[0273] In some embodiments, lipid nanoparticle formulations may
comprise 35 to 45% cationic lipid, 40% to 50% cationic lipid, 50%
to 60% cationic lipid and/or 55% to 65% cationic lipid. In some
embodiments, the ratio of lipid to RNA (e.g., mRNA) in lipid
nanoparticles may be 5:1 to 20:1, 10:1 to 25:1, 15:1 to 30:1 and/or
at least 30:1.
[0274] In some embodiments, the ratio of PEG in the lipid
nanoparticle formulations may be increased or decreased and/or the
carbon chain length of the PEG lipid may be modified from C14 to
C18 to alter the pharmacokinetics and/or biodistribution of the
lipid nanoparticle formulations. As a non-limiting example, lipid
nanoparticle formulations may contain 0.5% to 3.0%, 1.0% to 3.5%,
1.5% to 4.0%, 2.0% to 4.5%, 2.5% to 5.0% and/or 3.0% to 6.0% of the
lipid molar ratio of PEG-c-DOMG
(R-3-[.omega.-methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyl-
oxypropyl-3-amine) (also referred to herein as PEG-DOMG) as
compared to the cationic lipid, DSPC and cholesterol. In some
embodiments, the PEG-c-DOMG may be replaced with a PEG lipid such
as, but not limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol,
methoxypolyethylene glycol), PEG-DMG (1,2-Dimyristoyl-sn-glycerol)
and/or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene
glycol). The cationic lipid may be selected from any lipid known in
the art such as, but not limited to, DLin-MC3-DMA, DLin-DMA,
C12-200 and DLin-KC2-DMA.
[0275] In some embodiments, a bacterial RNA (e.g. mRNA) vaccine
formulation is a nanoparticle that comprises at least one lipid.
The lipid may be selected from, but is not limited to, DLin-DMA,
DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA,
PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids. In
some embodiments, the lipid may be a cationic lipid such as, but
not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA,
DODMA and amino alcohol lipids. The amino alcohol cationic lipid
may be the lipids described in and/or made by the methods described
in U.S. Patent Publication No. US20130150625, herein incorporated
by reference in its entirety. As a non-limiting example, the
cationic lipid may be
2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-
-dien-1-yloxy]methyl}propan-1-ol (Compound 1 in US20130150625);
2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methy-
l}propan-1-ol (Compound 2 in US20130150625);
2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propa-
n-1-ol (Compound 3 in US20130150625); and
2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-oc-
tadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (Compound 4 in
US20130150625); or any pharmaceutically acceptable salt or
stereoisomer thereof.
[0276] Lipid nanoparticle formulations typically comprise a lipid,
in particular, an ionizable cationic lipid, for example,
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), or
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and
further comprise a neutral lipid, a sterol and a molecule capable
of reducing particle aggregation, for example a PEG or PEG-modified
lipid.
[0277] In some embodiments, a lipid nanoparticle formulation
consists essentially of (i) at least one lipid selected from the
group consisting of
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) a
neutral lipid selected from DSPC, DPPC, POPC, DOPE and compounds of
formula I-IV; (iii) a sterol, e.g., cholesterol; and (iv) a
PEG-lipid, e.g., PEG-DMG or PEG-cDMA, in a molar ratio of 20-60%
cationic lipid: 5-25% neutral lipid: 25-55% sterol; 0.5-15%
PEG-lipid.
[0278] In some embodiments, a lipid nanoparticle formulation
includes 25% to 75% on a molar basis of a cationic lipid selected
from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane
(DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate
(DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g.,
35 to 65%, 45 to 65%, 60%, 57.5%, 50% or 40% on a molar basis.
[0279] In some embodiments, a lipid nanoparticle formulation
includes 0.5% to 15% on a molar basis of the neutral lipid, e.g., 3
to 12%, 5 to 10% or 15%, 10%, or 7.5% on a molar basis. Examples of
neutral lipids include, without limitation, DSPC, POPC, DPPC, DOPE
and compounds of formula I-IV. In some embodiments, the formulation
includes 5% to 50% on a molar basis of the sterol (e.g., 15 to 45%,
20 to 40%, 40%, 38.5%, 35%, or 31% on a molar basis. A non-limiting
example of a sterol is cholesterol. In some embodiments, a lipid
nanoparticle formulation includes 0.5% to 20% on a molar basis of
the PEG or PEG-modified lipid (e.g., 0.5 to 10%, 0.5 to 5%, 1.5%,
0.5%, 1.5%, 3.5%, or 5% on a molar basis. In some embodiments, a
PEG or PEG modified lipid comprises a PEG molecule of an average
molecular weight of 2,000 Da. In some embodiments, a PEG or PEG
modified lipid comprises a PEG molecule of an average molecular
weight of less than 2,000, for example around 1,500 Da, around
1,000 Da, or around 500 Da. Non-limiting examples of PEG-modified
lipids include PEG-distearoyl glycerol (PEG-DMG) (also referred
herein as PEG-C14 or C14-PEG), PEG-cDMA (further discussed in Reyes
et al. J. Controlled Release, 107, 276-287 (2005) the contents of
which are herein incorporated by reference in their entirety).
[0280] In some embodiments, lipid nanoparticle formulations include
25-75% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15%
of the neutral lipid, 5-50% of the sterol, and 0.5-20% of the PEG
or PEG-modified lipid on a molar basis.
[0281] In some embodiments, lipid nanoparticle formulations include
35-65% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12%
of the neutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG
or PEG-modified lipid on a molar basis.
[0282] In some embodiments, lipid nanoparticle formulations include
45-65% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10%
of the neutral lipid, 25-40% of the sterol, and 0.5-10% of the PEG
or PEG-modified lipid on a molar basis.
[0283] In some embodiments, lipid nanoparticle formulations include
60% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 7.5% of
the neutral lipid, 31% of the sterol, and 1.5% of the PEG or
PEG-modified lipid on a molar basis.
[0284] In some embodiments, lipid nanoparticle formulations include
50% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 10% of
the neutral lipid, 38.5% of the sterol, and 1.5% of the PEG or
PEG-modified lipid on a molar basis.
[0285] In some embodiments, lipid nanoparticle formulations include
50% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 10% of
the neutral lipid, 35% of the sterol, 4.5% or 5% of the PEG or
PEG-modified lipid, and 0.5% of the targeting lipid on a molar
basis.
[0286] In some embodiments, lipid nanoparticle formulations include
40% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 15% of
the neutral lipid, 40% of the sterol, and 5% of the PEG or
PEG-modified lipid on a molar basis.
[0287] In some embodiments, lipid nanoparticle formulations include
57.2% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 7.1% of
the neutral lipid, 34.3% of the sterol, and 1.4% of the PEG or
PEG-modified lipid on a molar basis.
[0288] In some embodiments, lipid nanoparticle formulations include
57.5% of a cationic lipid selected from the PEG lipid is PEG-cDMA
(PEG-cDMA is further discussed in Reyes et al. (J. Controlled
Release, 107, 276-287 (2005), the contents of which are herein
incorporated by reference in their entirety), 7.5% of the neutral
lipid, 31.5% of the sterol, and 3.5% of the PEG or PEG-modified
lipid on a molar basis.
[0289] In some embodiments, lipid nanoparticle formulations consist
essentially of a lipid mixture in molar ratios of 20-70% cationic
lipid: 5-45% neutral lipid: 20-55% cholesterol: 0.5-15%
PEG-modified lipid. In some embodiments, lipid nanoparticle
formulations consist essentially of a lipid mixture in a molar
ratio of 20-60% cationic lipid: 5-25% neutral lipid: 25-55%
cholesterol: 0.5-15% PEG-modified lipid.
[0290] In some embodiments, the molar lipid ratio is 50/10/38.5/1.5
(mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified
lipid, e.g., PEG-DMG, PEG-DSG or PEG-DPG), 57.2/7.1134.3/1.4 (mol %
cationic lipid/neutral lipid, e.g., DPPC/Chol/PEG-modified lipid,
e.g., PEG-cDMA), 40/15/40/5 (mol % cationic lipid/neutral lipid,
e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG),
50/10/35/4.5/0.5 (mol % cationic lipid/neutral lipid, e.g.,
DSPC/Chol/PEG-modified lipid, e.g., PEG-DSG), 50/10/35/5 (cationic
lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,
PEG-DMG), 40/10/40/10 (mol % cationic lipid/neutral lipid, e.g.,
DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA),
35/15/40/10 (mol % cationic lipid/neutral lipid, e.g.,
DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA) or
52/13/30/5 (mol % cationic lipid/neutral lipid, e.g.,
DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA).
[0291] Non-limiting examples of lipid nanoparticle compositions and
methods of making them are described, for example, in Semple et al.
(2010) Nat. Biotechnol. 28:172-176; Jayarama et al. (2012), Angew.
Chem. Int. Ed., 51: 8529-8533; and Maier et al. (2013) Molecular
Therapy 21, 1570-1578 (the contents of each of which are
incorporated herein by reference in their entirety).
[0292] In some embodiments, lipid nanoparticle formulations may
comprise a cationic lipid, a PEG lipid and a structural lipid and
optionally comprise a non-cationic lipid. As a non-limiting
example, a lipid nanoparticle may comprise 40-60% of cationic
lipid, 5-15% of a non-cationic lipid, 1-2% of a PEG lipid and
30-50% of a structural lipid. As another non-limiting example, the
lipid nanoparticle may comprise 50% cationic lipid, 10%
non-cationic lipid, 1.5% PEG lipid and 38.5% structural lipid. As
yet another non-limiting example, a lipid nanoparticle may comprise
55% cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid and
32.5% structural lipid. In some embodiments, the cationic lipid may
be any cationic lipid described herein such as, but not limited to,
DLin-KC2-DMA, DLin-MC3-DMA and L319.
[0293] In some embodiments, the lipid nanoparticle formulations
described herein may be 4 component lipid nanoparticles. The lipid
nanoparticle may comprise a cationic lipid, a non-cationic lipid, a
PEG lipid and a structural lipid. As a non-limiting example, the
lipid nanoparticle may comprise 40-60% of cationic lipid, 5-15% of
a non-cationic lipid, 1-2% of a PEG lipid and 30-50% of a
structural lipid. As another non-limiting example, the lipid
nanoparticle may comprise 50% cationic lipid, 10% non-cationic
lipid, 1.5% PEG lipid and 38.5% structural lipid. As yet another
non-limiting example, the lipid nanoparticle may comprise 55%
cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid and 32.5%
structural lipid. In some embodiments, the cationic lipid may be
any cationic lipid described herein such as, but not limited to,
DLin-KC2-DMA, DLin-MC3-DMA and L319.
[0294] In some embodiments, the lipid nanoparticle formulations
described herein may comprise a cationic lipid, a non-cationic
lipid, a PEG lipid and a structural lipid. As a non-limiting
example, the lipid nanoparticle comprises 50% of the cationic lipid
DLin-KC2-DMA, 10% of the non-cationic lipid DSPC, 1.5% of the PEG
lipid PEG-DOMG and 38.5% of the structural lipid cholesterol. As a
non-limiting example, the lipid nanoparticle comprises 50% of the
cationic lipid DLin-MC3-DMA, 10% of the non-cationic lipid DSPC,
1.5% of the PEG lipid PEG-DOMG and 38.5% of the structural lipid
cholesterol. As a non-limiting example, the lipid nanoparticle
comprises 50% of the cationic lipid DLin-MC3-DMA, 10% of the
non-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DMG and 38.5% of
the structural lipid cholesterol. As yet another non-limiting
example, the lipid nanoparticle comprises 55% of the cationic lipid
L319, 10% of the non-cationic lipid DSPC, 2.5% of the PEG lipid
PEG-DMG and 32.5% of the structural lipid cholesterol.
[0295] Relative amounts of the active ingredient, the
pharmaceutically acceptable excipient, and/or any additional
ingredients in a vaccine composition may vary, depending upon the
identity, size, and/or condition of the subject being treated and
further depending upon the route by which the composition is to be
administered. For example, the composition may comprise between
0.1% and 99% (w/w) of the active ingredient. By way of example, the
composition may comprise between 0.1% and 100%, e.g., between 0.5
and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active
ingredient.
[0296] In some embodiments, the bacterial RNA (e.g. mRNA) vaccine
composition may comprise the polynucleotide described herein,
formulated in a lipid nanoparticle comprising MC3, Cholesterol,
DSPC and PEG2000-DMG, the buffer trisodium citrate, sucrose and
water for injection. As a non-limiting example, the composition
comprises: 2.0 mg/mL of drug substance (e.g., polynucleotides
encoding H10N8 hMPV), 21.8 mg/mL of MC3, 10.1 mg/mL of cholesterol,
5.4 mg/mL of DSPC, 2.7 mg/mL of PEG2000-DMG, 5.16 mg/mL of
trisodium citrate, 71 mg/mL of sucrose and 1.0 mL of water for
injection.
[0297] In some embodiments, a nanoparticle (e.g., a lipid
nanoparticle) has a mean diameter of 10-500 nm, 20-400 nm, 30-300
nm, 40-200 nm. In some embodiments, a nanoparticle (e.g., a lipid
nanoparticle) has a mean diameter of 50-150 nm, 50-200 nm, 80-100
nm or 80-200 nm.
Liposomes, Lipoplexes, and Lipid Nanoparticles
[0298] The RNA (e.g., mRNA) vaccines of the disclosure can be
formulated using one or more liposomes, lipoplexes, or lipid
nanoparticles. In some embodiments, pharmaceutical compositions of
RNA (e.g., mRNA) vaccines include liposomes. Liposomes are
artificially-prepared vesicles which may primarily be composed of a
lipid bilayer and may be used as a delivery vehicle for the
administration of nutrients and pharmaceutical formulations.
Liposomes can be of different sizes such as, but not limited to, a
multilamellar vesicle (MLV) which may be hundreds of nanometers in
diameter and may contain a series of concentric bilayers separated
by narrow aqueous compartments, a small unicellular vesicle (SUV)
which may be smaller than 50 nm in diameter, and a large
unilamellar vesicle (LUV) which may be between 50 and 500 nm in
diameter. Liposome design may include, but is not limited to,
opsonins or ligands in order to improve the attachment of liposomes
to unhealthy tissue or to activate events such as, but not limited
to, endocytosis. Liposomes may contain a low or a high pH in order
to improve the delivery of the pharmaceutical formulations.
[0299] The formation of liposomes may depend on the physicochemical
characteristics such as, but not limited to, the pharmaceutical
formulation entrapped and the liposomal ingredients, the nature of
the medium in which the lipid vesicles are dispersed, the effective
concentration of the entrapped substance and its potential
toxicity, any additional processes involved during the application
and/or delivery of the vesicles, the optimization size,
polydispersity and the shelf-life of the vesicles for the intended
application, and the batch-to-batch reproducibility and possibility
of large-scale production of safe and efficient liposomal
products.
[0300] In some embodiments, pharmaceutical compositions described
herein may include, without limitation, liposomes such as those
formed from 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA)
liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.),
1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA),
2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane
(DLin-KC2-DMA), and MC3 (US20100324120; herein incorporated by
reference in its entirety) and liposomes which may deliver small
molecule drugs such as, but not limited to, DOXIL.RTM. from Janssen
Biotech, Inc. (Horsham, Pa.).
[0301] In some embodiments, pharmaceutical compositions described
herein may include, without limitation, liposomes such as those
formed from the synthesis of stabilized plasmid-lipid particles
(SPLP) or stabilized nucleic acid lipid particle (SNALP) that have
been previously described and shown to be suitable for
oligonucleotide delivery in vitro and in vivo (see Wheeler et al.
Gene Therapy. 1999 6:271-281; Zhang et al. Gene Therapy. 1999
6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et
al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al., Nature.
2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287;
Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J Clin
Invest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008
19:125-132; U.S. Patent Publication No US20130122104; all of which
are incorporated herein in their entireties). The original
manufacture method by Wheeler et al. was a detergent dialysis
method, which was later improved by Jeffs et al. and is referred to
as the spontaneous vesicle formation method. The liposome
formulations are composed of 3 to 4 lipid components in addition to
the polynucleotide. As an example a liposome can contain, but is
not limited to, 55% cholesterol, 20% disteroylphosphatidyl choline
(DSPC), 10% PEG-S-DSG, and 15%
1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by
Jeffs et al. As another example, certain liposome formulations may
contain, but are not limited to, 48% cholesterol, 20% DSPC, 2%
PEG-c-DMA, and 30% cationic lipid, where the cationic lipid can be
1,2-distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA,
or 1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as
described by Heyes et al.
[0302] In some embodiments, liposome formulations may comprise from
about 25.0% cholesterol to about 40.0% cholesterol, from about
30.0% cholesterol to about 45.0% cholesterol, from about 35.0%
cholesterol to about 50.0% cholesterol and/or from about 48.5%
cholesterol to about 60% cholesterol. In some embodiments,
formulations may comprise a percentage of cholesterol selected from
the group consisting of 28.5%, 31.5%, 33.5%, 36.5%, 37.0%, 38.5%,
39.0% and 43.5%. In some embodiments, formulations may comprise
from about 5.0% to about 10.0% DSPC and/or from about 7.0% to about
15.0% DSPC.
[0303] In some embodiments, the RNA (e.g., mRNA) vaccine
pharmaceutical compositions may be formulated in liposomes such as,
but not limited to, DiLa2 liposomes (Marina Biotech, Bothell,
Wash.), SMARTICLES.RTM. (Marina Biotech, Bothell, Wash.), neutral
DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes
(e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer
Biology & Therapy 2006 5(12)1708-1713); herein incorporated by
reference in its entirety) and hyaluronan-coated liposomes (Quiet
Therapeutics, Israel).
[0304] In some embodiments, the cationic lipid may be a low
molecular weight cationic lipid such as those described in U.S.
Patent Application No. 20130090372, the contents of which are
herein incorporated by reference in their entirety.
[0305] In some embodiments, the RNA (e.g., mRNA) vaccines may be
formulated in a lipid vesicle, which may have crosslinks between
functionalized lipid bilayers.
[0306] In some embodiments, the RNA (e.g., mRNA) vaccines may be
formulated in a lipid-polycation complex. The formation of the
lipid-polycation complex may be accomplished by methods known in
the art and/or as described in U.S. Pub. No. 20120178702, herein
incorporated by reference in its entirety. As a non-limiting
example, the polycation may include a cationic peptide or a
polypeptide such as, but not limited to, polylysine, polyornithine
and/or polyarginine. In some embodiments, the RNA (e.g., mRNA)
vaccines may be formulated in a lipid-polycation complex, which may
further include a non-cationic lipid such as, but not limited to,
cholesterol or dioleoyl phosphatidylethanolamine (DOPE).
[0307] In some embodiments, the ratio of PEG in the lipid
nanoparticle (LNP) formulations may be increased or decreased
and/or the carbon chain length of the PEG lipid may be modified
from C14 to C18 to alter the pharmacokinetics and/or
biodistribution of the LNP formulations. As a non-limiting example,
LNP formulations may contain from about 0.5% to about 3.0%, from
about 1.0% to about 3.5%, from about 1.5% to about 4.0%, from about
2.0% to about 4.5%, from about 2.5% to about 5.0% and/or from about
3.0% to about 6.0% of the lipid molar ratio of PEG-c-DOMG
(R-3-[.omega.-methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyl-
oxypropyl-3-amine) (also referred to herein as PEG-DOMG) as
compared to the cationic lipid, DSPC and cholesterol. In some
embodiments, the PEG-c-DOMG may be replaced with a PEG lipid such
as, but not limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol,
methoxypolyethylene glycol), PEG-DMG (1,2-Dimyristoyl-sn-glycerol)
and/or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene
glycol). The cationic lipid may be selected from any lipid known in
the art such as, but not limited to, DLin-MC3-DMA, DLin-DMA,
C12-200 and DLin-KC2-DMA.
[0308] In some embodiments, the RNA (e.g., mRNA) vaccines may be
formulated in a lipid nanoparticle.
[0309] In some embodiments, the RNA (e.g., mRNA) vaccine
formulation comprising the polynucleotide is a nanoparticle which
may comprise at least one lipid. The lipid may be selected from,
but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200,
DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated
lipids and amino alcohol lipids. In another aspect, the lipid may
be a cationic lipid such as, but not limited to, DLin-DMA,
DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol
lipids. The amino alcohol cationic lipid may be the lipids
described in and/or made by the methods described in U.S. Patent
Publication No. US20130150625, herein incorporated by reference in
its entirety. As a non-limiting example, the cationic lipid may be
2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-
-dien-1-yloxy]methyl}propan-1-ol (Compound 1 in US2013/0150625);
2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methy-
l}propan-1-ol (Compound 2 in US2013/0150625);
2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propa-
n-1-ol (Compound 3 in US2013/0150625); and
2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-oc-
tadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (Compound 4 in
US2013/0150625); or any pharmaceutically acceptable salt or
stereoisomer thereof.
[0310] Lipid nanoparticle formulations typically comprise a lipid,
in particular, an ionizable cationic lipid, for example,
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), or
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and
further comprise a neutral lipid, a sterol and a molecule capable
of reducing particle aggregation, for example a PEG or PEG-modified
lipid.
[0311] In some embodiments, the lipid nanoparticle formulation
consists essentially of (i) at least one lipid selected from the
group consisting of
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) a
neutral lipid selected from DSPC, DPPC, POPC, DOPE and compounds of
formula I-IV; (iii) a sterol, e.g., cholesterol; and (iv) a
PEG-lipid, e.g., PEG-DMG or PEG-cDMA, in a molar ratio of about
20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol; 0.5-15%
PEG-lipid.
[0312] In some embodiments, the formulation includes from about
5-25% to about 75% on a molar basis of a cationic lipid selected
from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane
(DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate
(DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g.,
from about 35 to about 65%, from about 45 to about 65%, about 60%,
about 57.5%, about 50% or about 40% on a molar basis.
[0313] In some embodiments, the formulation includes from about
0.5% to about 15% on a molar basis of the neutral lipid e.g., from
about 3 to about 12%, from about 5 to about 10% or about 15%, about
10%, or about 7.5% on a molar basis. Examples of neutral lipids
include, but are not limited to, DSPC, POPC, DPPC, DOPE and
compounds of formula I-IV. In some embodiments, the formulation
includes from about 5% to about 50% on a molar basis of the sterol
(e.g., about 15 to about 45%, about 20 to about 40%, about 40%,
about 38.5%, about 35%, or about 31% on a molar basis. An exemplary
sterol is cholesterol. In some embodiments, the formulation
includes from about 0.5% to about 20% on a molar basis of the PEG
or PEG-modified lipid (e.g., about 0.5 to about 10%, about 0.5 to
about 5%, about 1.5%, about 0.5%, about 1.5%, about 3.5%, or about
5% on a molar basis. In some embodiments, the PEG or PEG modified
lipid comprises a PEG molecule of an average molecular weight of
2,000 Da. In other embodiments, the PEG or PEG modified lipid
comprises a PEG molecule of an average molecular weight of less
than 2,000, for example around 1,500 Da, around 1,000 Da, or around
500 Da. Examples of PEG-modified lipids include, but are not
limited to, PEG-distearoyl glycerol (PEG-DMG) (also referred herein
as PEG-C14 or C14-PEG), PEG-cDMA (further discussed in Reyes et al.
J. Controlled Release, 107, 276-287 (2005) the contents of which
are herein incorporated by reference in their entirety)
[0314] In some embodiments, the formulations of the present
disclosure include 25-75% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15%
of the neutral lipid, 5-50% of the sterol, and 0.5-20% of the PEG
or PEG-modified lipid on a molar basis.
[0315] In some embodiments, the formulations of the present
disclosure include 35-65% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12%
of the neutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG
or PEG-modified lipid on a molar basis.
[0316] In some embodiments, the formulations of the present
disclosure include 45-65% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10%
of the neutral lipid, 25-40% of the sterol, and 0.5-10% of the PEG
or PEG-modified lipid on a molar basis.
[0317] In some embodiments, the formulations of the present
disclosure include about 60% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about
7.5% of the neutral lipid, about 31% of the sterol, and about 1.5%
of the PEG or PEG-modified lipid on a molar basis.
[0318] In some embodiments, the formulations of the present
disclosure include about 50% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about
10% of the neutral lipid, about 38.5% of the sterol, and about 1.5%
of the PEG or PEG-modified lipid on a molar basis.
[0319] In some embodiments, the formulations of the present
disclosure include about 50% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about
10% of the neutral lipid, about 35% of the sterol, about 4.5% or
about 5% of the PEG or PEG-modified lipid, and about 0.5% of the
targeting lipid on a molar basis.
[0320] In some embodiments, the formulations of the present
disclosure include about 40% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about
15% of the neutral lipid, about 40% of the sterol, and about 5% of
the PEG or PEG-modified lipid on a molar basis.
[0321] In some embodiments, the formulations of the present
disclosure include about 57.2% of a cationic lipid selected from
2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),
dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and
di((Z)-non-2-en-1-yl)
9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about
7.1% of the neutral lipid, about 34.3% of the sterol, and about
1.4% of the PEG or PEG-modified lipid on a molar basis.
[0322] In some embodiments, the formulations of the present
disclosure include about 57.5% of a cationic lipid selected from
the PEG lipid is PEG-cDMA (PEG-cDMA is further discussed in Reyes
et al. (J. Controlled Release, 107, 276-287 (2005), the contents of
which are herein incorporated by reference in their entirety),
about 7.5% of the neutral lipid, about 31.5% of the sterol, and
about 3.5% of the PEG or PEG-modified lipid on a molar basis.
[0323] In some embodiments, lipid nanoparticle formulation consists
essentially of a lipid mixture in molar ratios of about 20-70%
cationic lipid: 5-45% neutral lipid: 20-55% cholesterol: 0.5-15%
PEG-modified lipid; more preferably in a molar ratio of about
20-60% cationic lipid: 5-25% neutral lipid: 25-55% cholesterol:
0.5-15% PEG-modified lipid.
[0324] In some embodiments, the molar lipid ratio is approximately
50/10/38.5/1.5 (mol % cationic lipid/neutral lipid, e.g.,
DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG, PEG-DSG or PEG-DPG),
57.2/7.1134.3/1.4 (mol % cationic lipid/neutral lipid, e.g.,
DPPC/Chol/PEG-modified lipid, e.g., PEG-cDMA), 40/15/40/5 (mol %
cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid,
e.g., PEG-DMG), 50/10/35/4.5/0.5 (mol % cationic lipid/neutral
lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DSG),
50/10/35/5 (cationic lipid/neutral lipid, e.g.,
DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG), 40/10/40/10 (mol %
cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid,
e.g., PEG-DMG or PEG-cDMA), 35/15/40/10 (mol % cationic
lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,
PEG-DMG or PEG-cDMA) or 52/13/30/5 (mol % cationic lipid/neutral
lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or
PEG-cDMA).
[0325] Examples of lipid nanoparticle compositions and methods of
making same are described, for example, in Semple et al. (2010)
Nat. Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem.
Int. Ed., 51: 8529-8533; and Maier et al. (2013) Molecular Therapy
21, 1570-1578 (the contents of each of which are incorporated
herein by reference in their entirety).
[0326] In some embodiments, the lipid nanoparticle formulations
described herein may comprise a cationic lipid, a PEG lipid and a
structural lipid and optionally comprise a non-cationic lipid. As a
non-limiting example, the lipid nanoparticle may comprise about
40-60% of cationic lipid, about 5-15% of a non-cationic lipid,
about 1-2% of a PEG lipid and about 30-50% of a structural lipid.
As another non-limiting example, the lipid nanoparticle may
comprise about 50% cationic lipid, about 10% non-cationic lipid,
about 1.5% PEG lipid and about 38.5% structural lipid. As yet
another non-limiting example, the lipid nanoparticle may comprise
about 55% cationic lipid, about 10% non-cationic lipid, about 2.5%
PEG lipid and about 32.5% structural lipid. In some embodiments,
the cationic lipid may be any cationic lipid described herein such
as, but not limited to, DLin-KC2-DMA, DLin-MC3-DMA and L319.
[0327] In some embodiments, the lipid nanoparticle formulations
described herein may be 4 component lipid nanoparticles. The lipid
nanoparticle may comprise a cationic lipid, a non-cationic lipid, a
PEG lipid and a structural lipid. As a non-limiting example, the
lipid nanoparticle may comprise about 40-60% of cationic lipid,
about 5-15% of a non-cationic lipid, about 1-2% of a PEG lipid and
about 30-50% of a structural lipid. As another non-limiting
example, the lipid nanoparticle may comprise about 50% cationic
lipid, about 10% non-cationic lipid, about 1.5% PEG lipid and about
38.5% structural lipid. As yet another non-limiting example, the
lipid nanoparticle may comprise about 55% cationic lipid, about 10%
non-cationic lipid, about 2.5% PEG lipid and about 32.5% structural
lipid. In some embodiments, the cationic lipid may be any cationic
lipid described herein such as, but not limited to, DLin-KC2-DMA,
DLin-MC3-DMA and L319.
[0328] In some embodiments, the lipid nanoparticle formulations
described herein may comprise a cationic lipid, a non-cationic
lipid, a PEG lipid and a structural lipid. As a non-limiting
example, the lipid nanoparticle comprise about 50% of the cationic
lipid DLin-KC2-DMA, about 10% of the non-cationic lipid DSPC, about
1.5% of the PEG lipid PEG-DOMG and about 38.5% of the structural
lipid cholesterol. As a non-limiting example, the lipid
nanoparticle comprise about 50% of the cationic lipid DLin-MC3-DMA,
about 10% of the non-cationic lipid DSPC, about 1.5% of the PEG
lipid PEG-DOMG and about 38.5% of the structural lipid cholesterol.
As a non-limiting example, the lipid nanoparticle comprise about
50% of the cationic lipid DLin-MC3-DMA, about 10% of the
non-cationic lipid DSPC, about 1.5% of the PEG lipid PEG-DMG and
about 38.5% of the structural lipid cholesterol. As yet another
non-limiting example, the lipid nanoparticle comprise about 55% of
the cationic lipid L319, about 10% of the non-cationic lipid DSPC,
about 2.5% of the PEG lipid PEG-DMG and about 32.5% of the
structural lipid cholesterol.
[0329] As a non-limiting example, the cationic lipid may be
selected from (20Z,23Z)--N,N-dimethylnonacosa-20,23-dien-10-amine,
(17Z,20Z)--N,N-dimemylhexacosa-17,20-dien-9-amine,
(1Z,19Z)--N5N-dimethylpentacosa-1 6, 19-dien-8-amine,
(13Z,16Z)--N,N-dimethyldocosa-13,16-dien-5-amine,
(12Z,15Z)--N,N-dimethylhenicosa-12,15-dien-4-amine,
(14Z,17Z)--N,N-dimethyltricosa-14,17-dien-6-amine,
(15Z,18Z)--N,N-dimethyltetracosa-15,18-dien-7-amine,
(18Z,21Z)--N,N-dimethylheptacosa-18,21-dien-10-amine,
(15Z,18Z)--N,N-dimethyltetracosa-15,18-dien-5-amine,
(14Z,17Z)--N,N-dimethyltricosa-14,17-dien-4-amine,
(19Z,22Z)--N,N-dimeihyloctacosa-19,22-dien-9-amine,
(18Z,21Z)--N,N-dimethylheptacosa-18,21-dien-8-amine,
(17Z,20Z)--N,N-dimethylhexacosa-17,20-dien-7-amine,
(16Z,19Z)--N,N-dimethylpentacosa-16,19-dien-6-amine,
(22Z,25Z)--N,N-dimethylhentriaconta-22,25-dien-10-amine, (21
Z,24Z)--N,N-dimethyltriaconta-21,24-dien-9-amine,
(18Z)--N,N-dimetylheptacos-18-en-10-amine,
(17Z)--N,N-dimethylhexacos-17-en-9-amine,
(19Z,22Z)--N,N-dimethyloctacosa-19,22-dien-7-amine,
N,N-dimethylheptacosan-10-amine,
(20Z,23Z)--N-ethyl-N-methylnonacosa-20,23-dien-10-amine,
1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl] pyrrolidine,
(20Z)--N,N-dimethylheptacos-20-en-1 0-amine,
(15Z)--N,N-dimethyleptacos-15-en-1 0-amine,
(14Z)--N,N-dimethylnonacos-14-en-10-amine,
(17Z)--N,N-dimethylnonacos-17-en-10-amine,
(24Z)--N,N-dimethyltritriacont-24-en-10-amine,
(20Z)--N,N-dimethylnonacos-20-en-1 0-amine,
(22Z)--N,N-dimethylhentriacont-22-en-10-amine,
(16Z)--N,N-dimethylpentacos-16-en-8-amine,
(12Z,15Z)--N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine,
(13Z,16Z)--N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine,
N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl] eptadecan-8-amine,
1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine,
N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine,
N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine,
N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropy-
l]nonadecan-10-amine,
N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine,
N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine,
N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}
dodecan-1-amine,
1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine,
1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine,
N,N-dimethyl-1-[0S,2R)-2-octylcyclopropyl]pentadecan-8-amine,
R--N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propa-
n-2-amine,
S--N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octy-
loxy)propan-2-amine,
1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrr-
olidine,
(2S)--N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z-
)-oct-5-en-1-yloxy]propan-2-amine,
1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azet-
idine,
(2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-ylo-
xy]propan-2-amine,
(2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]pr-
opan-2-amine,
N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-
-amine,
N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-am-
ine;
(2S)--N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(o-
ctyloxy)propan-2-amine,
(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)pro-
pan-2-amine,
(2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylprop-
an-2-amine,
1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2--
amine,
1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)pr-
opan-2-amine,
(2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpro-
pan-2-amine,
(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amin-
e,
1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,
1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,
(2R)--N,N-dimethyl-H(1-metoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1--
yloxy]propan-2-amine,
(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-di-
en-1-yloxy]propan-2-amine,
N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]-
methyl}cyclopropyl]octyl}oxy)propan-2-amine,
N,N-dimethyl-1-{[8-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-am-
ine and (11E,20Z,23Z)--N,N-dimethylnonacosa-11,20,2-trien-10-amine
or a pharmaceutically acceptable salt or stereoisomer thereof.
[0330] In some embodiments, the LNP formulations of the RNA (e.g.,
mRNA) vaccines may contain PEG-c-DOMG at 3% lipid molar ratio. In
some embodiments, the LNP formulations of the RNA (e.g., mRNA)
vaccines may contain PEG-c-DOMG at 1.5% lipid molar ratio.
[0331] In some embodiments, the pharmaceutical compositions of the
RNA (e.g., mRNA) vaccines may include at least one of the PEGylated
lipids described in International Publication No. WO2012/099755,
the contents of which are herein incorporated by reference in their
entirety.
[0332] In some embodiments, the LNP formulation may contain PEG-DMG
2000
(1,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethylene
glycol)-2000). In some embodiments, the LNP formulation may contain
PEG-DMG 2000, a cationic lipid known in the art and at least one
other component. In some embodiments, the LNP formulation may
contain PEG-DMG 2000, a cationic lipid known in the art, DSPC and
cholesterol. As a non-limiting example, the LNP formulation may
contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol. As another
non-limiting example the LNP formulation may contain PEG-DMG 2000,
DLin-DMA, DSPC and cholesterol in a molar ratio of 2:40:10:48 (see
e.g., Geall et al., Nonviral delivery of self-amplifying RNA (e.g.,
mRNA) vaccines, PNAS 2012; PMID: 22908294, the contents of each of
which are herein incorporated by reference in their entirety).
[0333] The lipid nanoparticles described herein may be made in a
sterile environment.
[0334] In some embodiments, the LNP formulation may be formulated
in a nanoparticle such as a nucleic acid-lipid particle. As a
non-limiting example, the lipid particle may comprise one or more
active agents or therapeutic agents; one or more cationic lipids
comprising from about 50 mol % to about 85 mol % of the total lipid
present in the particle; one or more non-cationic lipids comprising
from about 13 mol % to about 49.5 mol % of the total lipid present
in the particle; and one or more conjugated lipids that inhibit
aggregation of particles comprising from about 0.5 mol % to about 2
mol % of the total lipid present in the particle.
[0335] The nanoparticle formulations may comprise a phosphate
conjugate. The phosphate conjugate may increase in vivo circulation
times and/or increase the targeted delivery of the nanoparticle. As
a non-limiting example, the phosphate conjugates may include a
compound of any one of the formulas described in International
Application No. WO2013/033438, the contents of which are herein
incorporated by reference in its entirety.
[0336] The nanoparticle formulation may comprise a polymer
conjugate. The polymer conjugate may be a water soluble conjugate.
The polymer conjugate may have a structure as described in U.S.
Patent Application No. 2013/0059360, the contents of which are
herein incorporated by reference in its entirety. In some
embodiments, polymer conjugates with the polynucleotides of the
present disclosure may be made using the methods and/or segmented
polymeric reagents described in U.S. Patent Application No.
2013/0072709, the contents of which are herein incorporated by
reference in its entirety. In some embodiments, the polymer
conjugate may have pendant side groups comprising ring moieties
such as, but not limited to, the polymer conjugates described in
U.S. Patent Publication No. US2013/0196948, the contents which are
herein incorporated by reference in its entirety.
[0337] The nanoparticle formulations may comprise a conjugate to
enhance the delivery of nanoparticles of the present disclosure in
a subject. Further, the conjugate may inhibit phagocytic clearance
of the nanoparticles in a subject. In one aspect, the conjugate may
be a "self" peptide designed from the human membrane protein CD47
(e.g., the "self" particles described by Rodriguez et al. (Science
2013 339, 971-975), herein incorporated by reference in its
entirety). As shown by Rodriguez et al., the self peptides delayed
macrophage-mediated clearance of nanoparticles which enhanced
delivery of the nanoparticles. In another aspect, the conjugate may
be the membrane protein CD47 (e.g., see Rodriguez et al. Science
2013 339, 971-975, herein incorporated by reference in its
entirety). Rodriguez et al. showed that, similarly to "self"
peptides, CD47 can increase the circulating particle ratio in a
subject as compared to scrambled peptides and PEG coated
nanoparticles.
[0338] In some embodiments, the RNA (e.g., mRNA) vaccines of the
present disclosure are formulated in nanoparticles which comprise a
conjugate to enhance the delivery of the nanoparticles of the
present disclosure in a subject. The conjugate may be the CD47
membrane or the conjugate may be derived from the CD47 membrane
protein, such as the "self" peptide described previously. In some
embodiments, the nanoparticle may comprise PEG and a conjugate of
CD47 or a derivative thereof. In some embodiments, the nanoparticle
may comprise both the "self" peptide described above and the
membrane protein CD47.
[0339] In some embodiments, the RNA (e.g., mRNA) vaccine
pharmaceutical compositions comprise the polynucleotides of the
present disclosure and a conjugate that may have a degradable
linkage. Non-limiting examples of conjugates include an aromatic
moiety comprising an ionizable hydrogen atom, a spacer moiety, and
a water-soluble polymer. As a non-limiting example, pharmaceutical
compositions comprising a conjugate with a degradable linkage and
methods for delivering such pharmaceutical compositions are
described in U.S. Patent Publication No. US2013/0184443, the
contents of which are herein incorporated by reference in their
entirety.
[0340] The nanoparticle formulations may be a carbohydrate
nanoparticle comprising a carbohydrate carrier and a RNA (e.g.,
mRNA) vaccine. As a non-limiting example, the carbohydrate carrier
may include, but is not limited to, an anhydride-modified
phytoglycogen or glycogen-type material, phtoglycogen octenyl
succinate, phytoglycogen beta-dextrin, anhydride-modified
phytoglycogen beta-dextrin. (See e.g., International Publication
No. WO2012/109121; the contents of which are herein incorporated by
reference in their entirety).
[0341] Nanoparticle formulations of the present disclosure may be
coated with a surfactant or polymer in order to improve the
delivery of the particle. In some embodiments, the nanoparticle may
be coated with a hydrophilic coating such as, but not limited to,
PEG coatings and/or coatings that have a neutral surface charge.
The hydrophilic coatings may help to deliver nanoparticles with
larger payloads such as, but not limited to, RNA (e.g., mRNA)
vaccines within the central nervous system. As a non-limiting
example nanoparticles comprising a hydrophilic coating and methods
of making such nanoparticles are described in U.S. Patent
Publication No. US2013/0183244, the contents of which are herein
incorporated by reference in their entirety.
[0342] In some embodiments, the lipid nanoparticles of the present
disclosure may be hydrophilic polymer particles. Non-limiting
examples of hydrophilic polymer particles and methods of making
hydrophilic polymer particles are described in U.S. Patent
Publication No. US2013/0210991, the contents of which are herein
incorporated by reference in their entirety.
[0343] In some embodiments, the lipid nanoparticles of the present
disclosure may be hydrophobic polymer particles.
[0344] Lipid nanoparticle formulations may be improved by replacing
the cationic lipid with a biodegradable cationic lipid which is
known as a rapidly eliminated lipid nanoparticle (reLNP). Ionizable
cationic lipids, such as, but not limited to, DLinDMA,
DLin-KC2-DMA, and DLin-MC3-DMA, have been shown to accumulate in
plasma and tissues over time and may be a potential source of
toxicity. The rapid metabolism of the rapidly eliminated lipids can
improve the tolerability and therapeutic index of the lipid
nanoparticles by an order of magnitude from a 1 mg/kg dose to a 10
mg/kg dose in rat. Inclusion of an enzymatically degraded ester
linkage can improve the degradation and metabolism profile of the
cationic component, while still maintaining the activity of the
reLNP formulation. The ester linkage can be internally located
within the lipid chain or it may be terminally located at the
terminal end of the lipid chain. The internal ester linkage may
replace any carbon in the lipid chain.
[0345] In some embodiments, the internal ester linkage may be
located on either side of the saturated carbon.
[0346] In some embodiments, an immune response may be elicited by
delivering a lipid nanoparticle which may include a nanospecies, a
polymer and an immunogen. (U.S. Publication No. 2012/0189700 and
International Publication No. WO2012/099805; each of which is
herein incorporated by reference in their entirety). The polymer
may encapsulate the nanospecies or partially encapsulate the
nanospecies. The immunogen may be a recombinant protein, a modified
RNA and/or a polynucleotide described herein. In some embodiments,
the lipid nanoparticle may be formulated for use in a vaccine such
as, but not limited to, against a pathogen.
[0347] Lipid nanoparticles may be engineered to alter the surface
properties of particles so the lipid nanoparticles may penetrate
the mucosal barrier. Mucus is located on mucosal tissue such as,
but not limited to, oral (e.g., the buccal and esophageal membranes
and tonsil tissue), ophthalmic, gastrointestinal (e.g., stomach,
small intestine, large intestine, colon, rectum), nasal,
respiratory (e.g., nasal, pharyngeal, tracheal and bronchial
membranes), genital (e.g., vaginal, cervical and urethral
membranes). Nanoparticles larger than 10-200 nm which are preferred
for higher drug encapsulation efficiency and the ability to provide
the sustained delivery of a wide array of drugs have been thought
to be too large to rapidly diffuse through mucosal barriers. Mucus
is continuously secreted, shed, discarded or digested and recycled
so most of the trapped particles may be removed from the mucosa
tissue within seconds or within a few hours. Large polymeric
nanoparticles (200 nm-500 nm in diameter) which have been coated
densely with a low molecular weight polyethylene glycol (PEG)
diffused through mucus only 4 to 6-fold lower than the same
particles diffusing in water (Lai et al. PNAS 2007 104(5):1482-487;
Lai et al. Adv Drug Deliv Rev. 2009 61(2): 158-171; each of which
is herein incorporated by reference in their entirety). The
transport of nanoparticles may be determined using rates of
permeation and/or fluorescent microscopy techniques including, but
not limited to, fluorescence recovery after photobleaching (FRAP)
and high resolution multiple particle tracking (MPT). As a
non-limiting example, compositions which can penetrate a mucosal
barrier may be made as described in U.S. Pat. No. 8,241,670 or
International Patent Publication No. WO2013/110028, the contents of
each of which are herein incorporated by reference in its
entirety.
[0348] The lipid nanoparticle engineered to penetrate mucus may
comprise a polymeric material (i.e. a polymeric core) and/or a
polymer-vitamin conjugate and/or a tri-block co-polymer. The
polymeric material may include, but is not limited to, polyamines,
polyethers, polyamides, polyesters, polycarbamates, polyureas,
polycarbonates, poly(styrenes), polyimides, polysulfones,
polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines,
polyisocyanates, polyacrylates, polymethacrylates,
polyacrylonitriles, and polyarylates. The polymeric material may be
biodegradable and/or biocompatible. Non-limiting examples of
biocompatible polymers are described in International Patent
Publication No. WO2013/116804, the contents of which are herein
incorporated by reference in their entirety. The polymeric material
may additionally be irradiated. As a non-limiting example, the
polymeric material may be gamma irradiated (see e.g., International
App. No. WO2012/082165, herein incorporated by reference in its
entirety). Non-limiting examples of specific polymers include
poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA),
poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic
acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA),
poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide)
(PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone),
poly(D,L-lactide-co-caprolactone-co-glycolide),
poly(D,L-lactide-co-PEO-co-D,L-lactide),
poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,
polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate
(HPMA), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy
acids), polyanhydrides, polyorthoesters, poly(ester amides),
polyamides, poly(ester ethers), polycarbonates, polyalkylenes such
as polyethylene and polypropylene, polyalkylene glycols such as
poly(ethylene glycol) (PEG), polyalkylene oxides (PEO),
polyalkylene terephthalates such as poly(ethylene terephthalate),
polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such
as poly(vinyl acetate), polyvinyl halides such as poly(vinyl
chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene
(PS), polyurethanes, derivatized celluloses such as alkyl
celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose
esters, nitro celluloses, hydroxypropylcellulose,
carboxymethylcellulose, polymers of acrylic acids, such as
poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate),
poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate),
poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate),
poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl
acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate),
poly(octadecyl acrylate) and copolymers and mixtures thereof,
polydioxanone and its copolymers, polyhydroxyalkanoates,
polypropylene fumarate, polyoxymethylene, poloxamers,
poly(ortho)esters, poly(butyric acid), poly(valeric acid),
poly(lactide-co-caprolactone), PEG-PLGA-PEG and trimethylene
carbonate, polyvinylpyrrolidone. The lipid nanoparticle may be
coated or associated with a co-polymer such as, but not limited to,
a block co-polymer (such as a branched polyether-polyamide block
copolymer described in International Publication No. WO2013/012476,
herein incorporated by reference in its entirety), and
(poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene
glycol)) triblock copolymer (see e.g., U.S. Publication
2012/0121718 and U.S. Publication 2010/0003337 and U.S. Pat. No.
8,263,665, the contents of each of which are herein incorporated by
reference in their entirety). The co-polymer may be a polymer that
is generally regarded as safe (GRAS) and the formation of the lipid
nanoparticle may be in such a way that no new chemical entities are
created. For example, the lipid nanoparticle may comprise
poloxamers coating PLGA nanoparticles without forming new chemical
entities which are still able to rapidly penetrate human mucus
(Yang et al. Angew. Chem. Int. Ed. 2011 50:2597-2600; the contents
of which are herein incorporated by reference in their entirety). A
non-limiting scalable method to produce nanoparticles which can
penetrate human mucus is described by Xu et al. (see, e.g., J
Control Release 2013, 170(2):279-86; the contents of which are
herein incorporated by reference in their entirety).
[0349] The vitamin of the polymer-vitamin conjugate may be vitamin
E. The vitamin portion of the conjugate may be substituted with
other suitable components such as, but not limited to, vitamin A,
vitamin E, other vitamins, cholesterol, a hydrophobic moiety, or a
hydrophobic component of other surfactants (e.g., sterol chains,
fatty acids, hydrocarbon chains and alkylene oxide chains).
[0350] The lipid nanoparticle engineered to penetrate mucus may
include surface altering agents such as, but not limited to,
polynucleotides, anionic proteins (e.g., bovine serum albumin),
surfactants (e.g., cationic surfactants such as for example
dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives
(e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin,
polyethylene glycol and poloxamer), mucolytic agents (e.g.,
N-acetylcysteine, mugwort, bromelain, papain, clerodendrum,
acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna,
ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin,
gelsolin, thymosin .beta.4 dornase alfa, neltenexine, erdosteine)
and various DNases including rhDNase. The surface altering agent
may be embedded or enmeshed in the particle's surface or disposed
(e.g., by coating, adsorption, covalent linkage, or other process)
on the surface of the lipid nanoparticle. See, e.g., U.S.
Publication 2010/0215580 and U.S. Publication 2008/0166414 and U.S.
Publication 2013/0164343; the contents of each of which are herein
incorporated by reference in their entirety).
[0351] In some embodiments, the mucus penetrating lipid
nanoparticles may comprise at least one polynucleotide described
herein. The polynucleotide may be encapsulated in the lipid
nanoparticle and/or disposed on the surface of the particle. The
polynucleotide may be covalently coupled to the lipid nanoparticle.
Formulations of mucus penetrating lipid nanoparticles may comprise
a plurality of nanoparticles. Further, the formulations may contain
particles which may interact with the mucus and alter the
structural and/or adhesive properties of the surrounding mucus to
decrease mucoadhesion, which may increase the delivery of the mucus
penetrating lipid nanoparticles to the mucosal tissue.
[0352] In some embodiments, the mucus penetrating lipid
nanoparticles may be a hypotonic formulation comprising a mucosal
penetration enhancing coating. The formulation may be hypotonice
for the epithelium to which it is being delivered. Non-limiting
examples of hypotonic formulations may be found in International
Patent Publication No. WO2013/110028, the contents of which are
herein incorporated by reference in their entirety.
[0353] In some embodiments, in order to enhance the delivery
through the mucosal barrier the RNA (e.g., mRNA) vaccine
formulation may comprise or be a hypotonic solution. Hypotonic
solutions were found to increase the rate at which mucoinert
particles such as, but not limited to, mucus-penetrating particles,
were able to reach the vaginal epithelial surface (see e.g., Ensign
et al. Biomaterials 2013 34(28):6922-9, the contents of which are
herein incorporated by reference in their entirety).
[0354] In some embodiments, the RNA (e.g., mRNA) vaccine is
formulated as a lipoplex, such as, without limitation, the
ATUPLEX.TM. system, the DACC system, the DBTC system and other
siRNA-lipoplex technology from Silence Therapeutics (London, United
Kingdom), STEMFECT.TM. from STEMGENT.RTM. (Cambridge, Mass.), and
polyethylenimine (PEI) or protamine-based targeted and non-targeted
delivery of nucleic acids acids (Aleku et al. Cancer Res. 2008
68:9788-9798; Strumberg et al. Int J Clin Pharmacol Ther 2012
50:76-78; Santel et al., Gene Ther 2006 13:1222-1234; Santel et
al., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol.
Ther. 2010 23:334-344; Kaufmann et al. Microvasc Res 2010
80:286-293 Weide et al. J Immunother. 2009 32:498-507; Weide et al.
J Immunother. 2008 31:180-188; Pascolo Expert Opin. Biol. Ther.
4:1285-1294; Fotin-Mleczek et al., 2011 J. Immunother. 34:1-15;
Song et al., Nature Biotechnol. 2005, 23:709-717; Peer et al., Proc
Natl Acad Sci USA. 2007 6; 104:4095-4100; deFougerolles Hum Gene
Ther. 2008 19:125-132, the contents of each of which are
incorporated herein by reference in their entirety).
[0355] In some embodiments, such formulations may also be
constructed or compositions altered such that they passively or
actively are directed to different cell types in vivo, including
but not limited to hepatocytes, immune cells, tumor cells,
endothelial cells, antigen presenting cells, and leukocytes (Akinc
et al. Mol Ther. 2010 18:1357-1364; Song et al., Nat Biotechnol.
2005 23:709-717; Judge et al., J Clin Invest. 2009 119:661-673;
Kaufmann et al., Microvasc Res 2010 80:286-293; Santel et al., Gene
Ther 2006 13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370;
Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334-344; Basha et
al., Mol. Ther. 2011 19:2186-2200; Fenske and Cullis, Expert Opin
Drug Deliv. 2008 5:25-44; Peer et al., Science. 2008 319:627-630;
Peer and Lieberman, Gene Ther. 2011 18:1127-1133, the contents of
each of which are incorporated herein by reference in their
entirety). One example of passive targeting of formulations to
liver cells includes the DLin-DMA, DLin-KC2-DMA and
DLin-MC3-DMA-based lipid nanoparticle formulations, which have been
shown to bind to apolipoprotein E and promote binding and uptake of
these formulations into hepatocytes in vivo (Akinc et al. Mol Ther.
2010 18:1357-1364, the contents of which are incorporated herein by
reference in their entirety). Formulations can also be selectively
targeted through expression of different ligands on their surface
as exemplified by, but not limited by, folate, transferrin,
N-acetylgalactosamine (GalNAc), and antibody targeted approaches
(Kolhatkar et al., Curr Drug Discov Technol. 2011 8:197-206;
Musacchio and Torchilin, Front Biosci. 2011 16:1388-1412; Yu et
al., Mol Membr Biol. 2010 27:286-298; Patil et al., Crit Rev Ther
Drug Carrier Syst. 2008 25:1-61; Benoit et al., Biomacromolecules.
2011 12:2708-2714; Zhao et al., Expert Opin Drug Deliv. 2008
5:309-319; Akinc et al., Mol Ther. 2010 18:1357-1364; Srinivasan et
al., Methods Mol Biol. 2012 820:105-116; Ben-Arie et al., Methods
Mol Biol. 2012 757:497-507; Peer 2010 J Control Release. 20:63-68;
Peer et al., Proc Natl Acad Sci USA. 2007 104:4095-4100; Kim et
al., Methods Mol Biol. 2011 721:339-353; Subramanya et al., Mol
Ther. 2010 18:2028-2037; Song et al., Nat Biotechnol. 2005
23:709-717; Peer et al., Science. 2008 319:627-630; Peer and
Lieberman, Gene Ther. 2011 18:1127-1133, the contents of each of
which are incorporated herein by reference in their entirety).
[0356] In some embodiments, the RNA (e.g., mRNA) vaccine is
formulated as a solid lipid nanoparticle. A solid lipid
nanoparticle (SLN) may be spherical with an average diameter
between 10 to 1000 nm. SLN possess a solid lipid core matrix that
can solubilize lipophilic molecules and may be stabilized with
surfactants and/or emulsifiers. In some embodiments, the lipid
nanoparticle may be a self-assembly lipid-polymer nanoparticle (see
Zhang et al., ACS Nano, 2008, 2 (8), pp 1696-1702; the contents of
which are herein incorporated by reference in their entirety). As a
non-limiting example, the SLN may be the SLN described in
International Patent Publication No. WO2013/105101, the contents of
which are herein incorporated by reference in their entirety. As
another non-limiting example, the SLN may be made by the methods or
processes described in International Patent Publication No.
WO2013/105101, the contents of which are herein incorporated by
reference in their entirety.
[0357] Liposomes, lipoplexes, or lipid nanoparticles may be used to
improve the efficacy of polynucleotides directed protein production
as these formulations may be able to increase cell transfection by
the RNA (e.g., mRNA) vaccine; and/or increase the translation of
encoded protein. One such example involves the use of lipid
encapsulation to enable the effective systemic delivery of polyplex
plasmid DNA (Heyes et al., Mol Ther. 2007 15:713-720; the contents
of which are incorporated herein by reference in their entirety).
The liposomes, lipoplexes, or lipid nanoparticles may also be used
to increase the stability of the polynucleotide.
[0358] In some embodiments, the RNA (e.g., mRNA) vaccines of the
present disclosure can be formulated for controlled release and/or
targeted delivery. As used herein, "controlled release" refers to a
pharmaceutical composition or compound release profile that
conforms to a particular pattern of release to effect a therapeutic
outcome. In some embodiments, the RNA (e.g., mRNA) vaccines may be
encapsulated into a delivery agent described herein and/or known in
the art for controlled release and/or targeted delivery. As used
herein, the term "encapsulate" means to enclose, surround or
encase. As it relates to the formulation of the compounds of the
disclosure, encapsulation may be substantial, complete or partial.
The term "substantially encapsulated" means that at least greater
than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or
greater than 99.999% of the pharmaceutical composition or compound
of the disclosure may be enclosed, surrounded or encased within the
delivery agent. "Partially encapsulation" means that less than 10,
10, 20, 30, 40 50 or less of the pharmaceutical composition or
compound of the disclosure may be enclosed, surrounded or encased
within the delivery agent. Advantageously, encapsulation may be
determined by measuring the escape or the activity of the
pharmaceutical composition or compound of the disclosure using
fluorescence and/or electron micrograph. For example, at least 1,
5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99,
99.9, 99.99 or greater than 99.99% of the pharmaceutical
composition or compound of the disclosure are encapsulated in the
delivery agent.
[0359] In some embodiments, the controlled release formulation may
include, but is not limited to, tri-block co-polymers. As a
non-limiting example, the formulation may include two different
types of tri-block co-polymers (International Pub. No.
WO2012/131104 and WO2012/131106, the contents of each of which are
incorporated herein by reference in their entirety).
[0360] In some embodiments, the RNA (e.g., mRNA) vaccines may be
encapsulated into a lipid nanoparticle or a rapidly eliminated
lipid nanoparticle and the lipid nanoparticles or a rapidly
eliminated lipid nanoparticle may then be encapsulated into a
polymer, hydrogel and/or surgical sealant described herein and/or
known in the art. As a non-limiting example, the polymer, hydrogel
or surgical sealant may be PLGA, ethylene vinyl acetate (EVAc),
poloxamer, GELSITE.RTM. (Nanotherapeutics, Inc. Alachua, Fla.),
HYLENEX.RTM. (Halozyme Therapeutics, San Diego Calif.), surgical
sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, Ga.),
TISSELL.RTM. (Baxter International, Inc Deerfield, Ill.), PEG-based
sealants, and COSEAL.RTM. (Baxter International, Inc Deerfield,
Ill.).
[0361] In some embodiments, the lipid nanoparticle may be
encapsulated into any polymer known in the art which may form a gel
when injected into a subject. As another non-limiting example, the
lipid nanoparticle may be encapsulated into a polymer matrix which
may be biodegradable.
[0362] In some embodiments, the RNA (e.g., mRNA) vaccine
formulation for controlled release and/or targeted delivery may
also include at least one controlled release coating. Controlled
release coatings include, but are not limited to, OPADRY.RTM.,
polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone,
hydroxypropyl methylcellulose, hydroxypropyl cellulose,
hydroxyethyl cellulose, EUDRAGIT RL.RTM., EUDRAGIT RS.RTM. and
cellulose derivatives such as ethylcellulose aqueous dispersions
(AQUACOAT.RTM. and SURELEASE.RTM.).
[0363] In some embodiments, the RNA (e.g., mRNA) vaccine controlled
release and/or targeted delivery formulation may comprise at least
one degradable polyester which may contain polycationic side
chains. Degradeable polyesters include, but are not limited to,
poly(serine ester), poly(L-lactide-co-L-lysine),
poly(4-hydroxy-L-proline ester), and combinations thereof. In some
embodiments, the degradable polyesters may include a PEG
conjugation to form a PEGylated polymer.
[0364] In some embodiments, the RNA (e.g., mRNA) vaccine controlled
release and/or targeted delivery formulation comprising at least
one polynucleotide may comprise at least one PEG and/or PEG related
polymer derivatives as described in U.S. Pat. No. 8,404,222, the
contents of which are incorporated herein by reference in their
entirety.
[0365] In some embodiments, the RNA (e.g., mRNA) vaccine controlled
release delivery formulation comprising at least one polynucleotide
may be the controlled release polymer system described in
US2013/0130348, the contents of which are incorporated herein by
reference in their entirety.
[0366] In some embodiments, the RNA (e.g., mRNA) vaccines of the
present disclosure may be encapsulated in a therapeutic
nanoparticle, referred to herein as "therapeutic nanoparticle RNA
(e.g., mRNA) vaccines." Therapeutic nanoparticles may be formulated
by methods described herein and known in the art such as, but not
limited to, International Pub Nos. WO2010/005740, WO2010/030763,
WO2010/005721, WO2010/005723, WO2012/054923, U.S. Publication Nos.
US2011/0262491, US2010/0104645, US2010/0087337, US2010/0068285,
US2011/0274759, US2010/0068286, US2012/0288541, US2013/0123351 and
US2013/0230567 and U.S. Pat. Nos. 8,206,747, 8,293,276, 8,318,208
and 8,318,211; the contents of each of which are herein
incorporated by reference in their entirety. In some embodiments,
therapeutic polymer nanoparticles may be identified by the methods
described in US Pub No. US2012/0140790, the contents of which are
herein incorporated by reference in their entirety.
[0367] In some embodiments, the therapeutic nanoparticle RNA (e.g.,
mRNA) vaccine may be formulated for sustained release. As used
herein, "sustained release" refers to a pharmaceutical composition
or compound that conforms to a release rate over a specific period
of time. The period of time may include, but is not limited to,
hours, days, weeks, months and years. As a non-limiting example,
the sustained release nanoparticle may comprise a polymer and a
therapeutic agent such as, but not limited to, the polynucleotides
of the present disclosure (see International Pub No. WO2010/075072
and US Pub Nos. US2010/0216804, US2011/0217377 and US2012/0201859,
the contents of each of which are incorporated herein by reference
in their entirety). In another non-limiting example, the sustained
release formulation may comprise agents which permit persistent
bioavailability such as, but not limited to, crystals,
macromolecular gels and/or particulate suspensions (see U.S. Patent
Publication No US2013/0150295, the contents of each of which are
incorporated herein by reference in their entirety).
[0368] In some embodiments, the therapeutic nanoparticle RNA (e.g.,
mRNA) vaccines may be formulated to be target specific. As a
non-limiting example, the therapeutic nanoparticles may include a
corticosteroid (see International Pub. No. WO2011/084518, the
contents of which are incorporated herein by reference in their
entirety). As a non-limiting example, the therapeutic nanoparticles
may be formulated in nanoparticles described in International Pub
No. WO2008/121949, WO2010/005726, WO2010/005725, WO2011/084521 and
US Pub No. US2010/0069426, US2012/0004293 and US2010/0104655, the
contents of each of which are incorporated herein by reference in
their entirety.
[0369] In some embodiments, a subset of compounds of Formula (I)
includes those of Formula (IIa), (IIb), (IIc), or (IIe):
##STR00001##
[0370] or a salt or isomer thereof, wherein R.sub.4 is as described
herein.
[0371] In some embodiments, a subset of compounds of Formula (I)
includes those of Formula (IId):
##STR00002##
[0372] or a salt or isomer thereof, wherein n is 2, 3, or 4; and m,
R', R'', and R.sub.2 through R.sub.6 are as described herein. For
example, each of R.sub.2 and R.sub.3 may be independently selected
from the group consisting of C.sub.5-14 alkyl and C.sub.5-14
alkenyl.
[0373] In some embodiments, an ionizable cationic lipid of the
disclosure comprises a compound having structure:
##STR00003##
[0374] In some embodiments, an ionizable cationic lipid of the
disclosure comprises a compound having structure:
##STR00004##
[0375] In some embodiments, a non-cationic lipid of the disclosure
comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),
1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC),
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC),
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),
1,2-di-0-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),
1-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine
(OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),
1,2-dilinolenoyl-sn-glycero-3-phosphocholine,
1,2-diarachidonoyl-sn-glycero-3-phosphocholine,
1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,
1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE),
1,2-distearoyl-sn-glycero-3-phosphoethanolamine,
1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,
1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,
1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,
1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,
1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt
(DOPG), sphingomyelin, and mixtures thereof.
[0376] In some embodiments, a PEG modified lipid of the disclosure
comprises a PEG-modified phosphatidylethanolamine, a PEG-modified
phosphatidic acid, a PEG-modified ceramide, a PEG-modified
dialkylamine, a PEG-modified diacylglycerol, a PEG-modified
dialkylglycerol, and mixtures thereof. In some embodiments, the
PEG-modified lipid is PEG-DMG, PEG-c-DOMG (also referred to as
PEG-DOMG), PEG-DSG and/or PEG-DPG.
[0377] In some embodiments, a sterol of the disclosure comprises
cholesterol, fecosterol, sitosterol, ergosterol, campesterol,
stigmasterol, bras sicasterol, tomatidine, ursolic acid,
alpha-tocopherol, and mixtures thereof.
[0378] In some embodiments, a LNP of the disclosure comprises an
ionizable cationic lipid of Compound 1, wherein the non-cationic
lipid is DSPC, the structural lipid that is cholesterol, and the
PEG lipid is PEG-DMG.
[0379] In some embodiments, a LNP of the disclosure comprises an
N:P ratio of from about 2:1 to about 30:1.
[0380] In some embodiments, a LNP of the disclosure comprises an
N:P ratio of about 6:1.
[0381] In some embodiments, a LNP of the disclosure comprises an
N:P ratio of about 3:1.
[0382] In some embodiments, a LNP of the disclosure comprises a
wt/wt ratio of the ionizable cationic lipid component to the RNA of
from about 10:1 to about 100:1.
[0383] In some embodiments, a LNP of the disclosure comprises a
wt/wt ratio of the ionizable cationic lipid component to the RNA of
about 20:1.
[0384] In some embodiments, a LNP of the disclosure comprises a
wt/wt ratio of the ionizable cationic lipid component to the RNA of
about 10:1.
[0385] In some embodiments, a LNP of the disclosure has a mean
diameter from about 50 nm to about 150 nm.
[0386] In some embodiments, a LNP of the disclosure has a mean
diameter from about 70 nm to about 120 nm.
Pharmaceutical Formulations
[0387] Provided herein are compositions (e.g., pharmaceutical
compositions), methods, kits and reagents for prevention or
treatment of bacterial infection(s) in humans and other mammals,
for example. Bacterial RNA (e.g., mRNA) vaccines can be used as
therapeutic or prophylactic agents. They may be used in medicine to
prevent and/or treat infectious disease.
[0388] The term "pharmaceutical composition" refers to the
combination of an active agent with a carrier, inert or active,
making the composition especially suitable for diagnostic or
therapeutic use in vivo or ex vivo. A "pharmaceutically acceptable
carrier," after administered to or upon a subject, does not cause
undesirable physiological effects. The carrier in the
pharmaceutical composition must be "acceptable" also in the sense
that it is compatible with the active ingredient and can be capable
of stabilizing it. One or more solubilizing agents can be utilized
as pharmaceutical carriers for delivery of an active agent.
Examples of a pharmaceutically acceptable carrier include, but are
not limited to, biocompatible vehicles, adjuvants, additives, and
diluents to achieve a composition usable as a dosage form. Examples
of other carriers include colloidal silicon oxide, magnesium
stearate, cellulose, and sodium lauryl sulfate. Additional suitable
pharmaceutical carriers and diluents, as well as pharmaceutical
necessities for their use, are described in Remington's
Pharmaceutical Sciences.
[0389] In some embodiments, the disclosure features a
pharmaceutical composition comprising a nanoparticle composition
according to the preceding embodiments and a pharmaceutically
acceptable carrier. For example, the pharmaceutical composition is
refrigerated or frozen for storage and/or shipment (e.g., being
stored at a temperature of 4.degree. C. or lower, such as a
temperature between about -150.degree. C. and about 0.degree. C. or
between about -80.degree. C. and about -20.degree. C. (e.g., about
-5.degree. C., -10.degree. C., -15.degree. C., -20.degree. C.,
-25.degree. C., -30.degree. C., -40.degree. C., -50.degree. C.,
-60.degree. C., -70.degree. C., -80.degree. C., -90.degree. C.,
-130.degree. C. or -150.degree. C.). For example, the
pharmaceutical composition is a solution that is refrigerated for
storage and/or shipment at, for example, about -20.degree. C.,
-30.degree. C., -40.degree. C., -50.degree. C., -60.degree. C.,
-70.degree. C., or -80.degree. C.
[0390] In some embodiments, the disclosure provides a method of
delivering a therapeutic and/or prophylactic (e.g., RNA, such as
mRNA) to a cell (e.g., a mammalian cell). This method includes the
step of administering to a subject (e.g., a mammal, such as a
human) a nanoparticle composition including (i) a lipid component
including a phospholipid (such as a polyunsaturated lipid), a PEG
lipid, a structural lipid, and a compound of Formula (I), (IA),
(II), (IIa), (IIb), (IIc), (IId) or (IIe) and (ii) a therapeutic
and/or prophylactic, in which administering involves contacting the
cell with the nanoparticle composition, whereby the therapeutic
and/or prophylactic is delivered to the cell.
[0391] In some embodiments, the disclosure provides a method of
producing a polypeptide of interest in a cell (e.g., a mammalian
cell). The method includes the step of contacting the cell with a
nanoparticle composition including (i) a lipid component including
a phospholipid (such as a polyunsaturated lipid), a PEG lipid, a
structural lipid, and a compound of Formula (I), (IA), (II), (IIa),
(IIb), (IIc), (IId) or (IIe) and (ii) an mRNA encoding the
polypeptide of interest, whereby the mRNA is capable of being
translated in the cell to produce the polypeptide.
[0392] In some embodiments, the disclosure provides a method of
treating a disease or disorder in a mammal (e.g., a human) in need
thereof. The method includes the step of administering to the
mammal a therapeutically effective amount of a nanoparticle
composition including (i) a lipid component including a
phospholipid (such as a polyunsaturated lipid), a PEG lipid, a
structural lipid, and a compound of Formula (I), (IA), (II), (IIa),
(IIb), (IIc), (IId) or (IIe) and (ii) a therapeutic and/or
prophylactic (e.g., an mRNA). In some embodiments, the disease or
disorder is characterized by dysfunctional or aberrant protein or
polypeptide activity. For example, the disease or disorder is an
infectious disease such as a bacterial infection.
[0393] An "effective amount" of a bacterial vaccine is based, at
least in part, on the target tissue, target cell type, means of
administration, physical characteristics of the RNA (e.g., length,
nucleotide composition, and/or extent of modified nucleosides),
other components of the vaccine, and other determinants, such as
age, body weight, height, sex and general health of the subject.
Typically, an effective amount of bacterial vaccine provides an
induced or boosted immune response as a function of antigen
production in the cells of the subject. In some embodiments, an
effective amount of the bacterial RNA vaccine containing RNA
polynucleotides having at least one chemical modifications are more
efficient than a composition containing a corresponding unmodified
polynucleotide encoding the same antigen or a peptide antigen.
Increased antigen production may be demonstrated by increased cell
transfection (the percentage of cells transfected with the RNA
vaccine), increased protein translation and/or expression from the
polynucleotide, decreased nucleic acid degradation (as
demonstrated, for example, by increased duration of protein
translation from a modified polynucleotide), or altered antigen
specific immune response of the host cell.
[0394] In some embodiments, the disclosure provides a method of
delivering (e.g., specifically delivering) a therapeutic and/or
prophylactic to a mammalian organ (e.g., a liver, spleen, lung, or
femur). This method includes the step of administering to a subject
(e.g., a mammal) a nanoparticle composition including (i) a lipid
component including a phospholipid, a PEG lipid, a structural
lipid, and a compound of Formula (I), (IA), (II), (IIa), (IIb),
(IIc), (IId) or (IIe) and (ii) a therapeutic and/or prophylactic
(e.g., an mRNA), in which administering involves contacting the
cell with the nanoparticle composition, whereby the therapeutic
and/or prophylactic is delivered to the target organ (e.g., a
liver, spleen, lung, or femur).
[0395] In some embodiments, the disclosure features a method for
the enhanced delivery of a therapeutic and/or prophylactic (e.g.,
an mRNA) to a target tissue (e.g., a liver, spleen, lung, or
femur). This method includes administering to a subject (e.g., a
mammal) a nanoparticle composition, the composition including (i) a
lipid component including a compound of Formula (I), (IA), (II),
(IIa), (IIb), (IIc), (IId) or (IIe), a phospholipid, a structural
lipid, and a PEG lipid; and (ii) a therapeutic and/or prophylactic,
the administering including contacting the target tissue with the
nanoparticle composition, whereby the therapeutic and/or
prophylactic is delivered to the target tissue.
[0396] In some embodiments, the disclosure features a method of
lowering immunogenicity comprising introducing the nanoparticle
composition of the disclosure into cells, wherein the nanoparticle
composition reduces the induction of the cellular immune response
of the cells to the nanoparticle composition, as compared to the
induction of the cellular immune response in cells induced by a
reference composition which comprises a reference lipid instead of
a compound of Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId)
or (IIe). For example, the cellular immune response is an innate
immune response, an adaptive immune response, or both.
[0397] The disclosure also includes methods of synthesizing a
compound of Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or
(IIe) and methods of making a nanoparticle composition including a
lipid component comprising the compound of Formula (I), (IA), (II),
(IIa), (IIb), (IIc), (IId) or (IIe).
Modes of Vaccine Administration
[0398] Bacterial RNA (e.g. mRNA) vaccines may be administered by
any route which results in a therapeutically effective outcome.
These include, but are not limited, to intradermal, intramuscular,
and/or subcutaneous administration. The present disclosure provides
methods comprising administering RNA (e.g., mRNA) vaccines to a
subject in need thereof. The exact amount required will vary from
subject to subject, depending on the species, age, and general
condition of the subject, the severity of the disease, the
particular composition, its mode of administration, its mode of
activity, and the like. Bacterial RNA (e.g., mRNA) vaccines
compositions are typically formulated in dosage unit form for ease
of administration and uniformity of dosage. It will be understood,
however, that the total daily usage of RNA (e.g., mRNA) vaccine
compositions may be decided by the attending physician within the
scope of sound medical judgment. The specific therapeutically
effective, prophylactically effective, or appropriate imaging dose
level for any particular patient will depend upon a variety of
factors including the disorder being treated and the severity of
the disorder; the activity of the specific compound employed; the
specific composition employed; the age, body weight, general
health, sex and diet of the patient; the time of administration,
route of administration, and rate of excretion of the specific
compound employed; the duration of the treatment; drugs used in
combination or coincidental with the specific compound employed;
and like factors well known in the medical arts.
[0399] Bacterial RNA (e.g., mRNA) vaccines may be administered with
other prophylactic or therapeutic compounds. As a non-limiting
example, a prophylactic or therapeutic compound may be an adjuvant
or a booster. As used herein, when referring to a prophylactic
composition, such as a vaccine, the term "booster" refers to an
extra administration of the prophylactic (vaccine) composition. A
booster (or booster vaccine) may be given after an earlier
administration of the prophylactic composition. The time of
administration between the initial administration of the
prophylactic composition and the booster may be, but is not limited
to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6
minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes,
20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55
minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7
hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14
hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours,
21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4
days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2
months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months,
9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3
years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10
years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years,
17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35
years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years,
70 years, 75 years, 80 years, 85 years, 90 years, 95 years or more
than 99 years. In exemplary embodiments, the time of administration
between the initial administration of the prophylactic composition
and the booster may be, but is not limited to, 1 week, 2 weeks, 3
weeks, 1 month, 2 months, 3 months, 6 months or 1 year.
[0400] In some embodiments, bacterial RNA vaccines may be
administered intramuscularly, intranasally or intradermally,
similarly to the administration of inactivated vaccines known in
the art.
[0401] The bacterial RNA vaccines may be utilized in various
settings depending on the prevalence of the infection or the degree
or level of unmet medical need. As a non-limiting example, the RNA
vaccines may be utilized to treat and/or prevent a variety of
infectious disease. RNA vaccines have superior properties in that
they produce much larger antibody titers, better neutralizing
immunity, produce more durable immune responses, and/or produce
responses earlier than commercially available vaccines.
[0402] Provided herein are pharmaceutical compositions including
bacterial RNA vaccines and RNA vaccine compositions and/or
complexes optionally in combination with one or more
pharmaceutically acceptable excipients.
[0403] Bacterial RNA (e.g., mRNA) vaccines may be formulated or
administered alone or in conjunction with one or more other
components. For instance, bacterial RNA vaccines (vaccine
compositions) may comprise other components including, but not
limited to, adjuvants.
[0404] In some embodiments, bacterial RNA vaccines do not include
an adjuvant (they are adjuvant free).
[0405] Bacterial RNA (e.g., mRNA) vaccines may be formulated or
administered in combination with one or more
pharmaceutically-acceptable excipients. In some embodiments,
vaccine compositions comprise at least one additional active
substances, such as, for example, a therapeutically-active
substance, a prophylactically-active substance, or a combination of
both. Vaccine compositions may be sterile, pyrogen-free or both
sterile and pyrogen-free. General considerations in the formulation
and/or manufacture of pharmaceutical agents, such as vaccine
compositions, may be found, for example, in Remington: The Science
and Practice of Pharmacy 21st ed., Lippincott Williams &
Wilkins, 2005 (incorporated herein by reference in its
entirety).
[0406] Formulations of the vaccine compositions described herein
may be prepared by any method known or hereafter developed in the
art of pharmacology. In general, such preparatory methods include
the step of bringing the active ingredient (e.g., mRNA
polynucleotide) into association with an excipient and/or one or
more other accessory ingredients, and then, if necessary and/or
desirable, dividing, shaping and/or packaging the product into a
desired single- or multi-dose unit.
[0407] Relative amounts of the active ingredient, the
pharmaceutically acceptable excipient, and/or any additional
ingredients in a pharmaceutical composition in accordance with the
disclosure will vary, depending upon the identity, size, and/or
condition of the subject treated and further depending upon the
route by which the composition is to be administered. By way of
example, the composition may comprise between 0.1% and 100%, e.g.,
between 0.5 and 50%, between 1-30%, between 5-80%, at least 80%
(w/w) active ingredient.
[0408] In some embodiments, bacterial RNA vaccines are formulated
using one or more excipients to: (1) increase stability; (2)
increase cell transfection; (3) permit the sustained or delayed
release (e.g., from a depot formulation); (4) alter the
biodistribution (e.g., target to specific tissues or cell types);
(5) increase the translation of encoded protein in vivo; and/or (6)
alter the release profile of encoded protein (antigen) in vivo. In
addition to traditional excipients such as any and all solvents,
dispersion media, diluents, or other liquid vehicles, dispersion or
suspension aids, surface active agents, isotonic agents, thickening
or emulsifying agents, preservatives, excipients can include,
without limitation, lipidoids, liposomes, lipid nanoparticles,
polymers, lipoplexes, core-shell nanoparticles, peptides, proteins,
cells transfected with bacterial RNA vaccines (e.g., for
transplantation into a subject), hyaluronidase, nanoparticle mimics
and combinations thereof.
Dosing/Administration
[0409] Provided herein are compositions (e.g., pharmaceutical
compositions), methods, kits and reagents for prevention and/or
treatment of bacterial infections in humans and other mammals.
Bacterial RNA vaccines can be used as therapeutic or prophylactic
agents. In some aspects, the RNA vaccines of the disclosure are
used to provide prophylactic protection from bacterial infections.
In some aspects, the RNA vaccines of the disclosure are used to
treat a bacterial infection. In some embodiments, the bacterial
vaccines of the present disclosure are used in the priming of
immune effector cells, for example, to activate peripheral blood
mononuclear cells (PBMCs) ex vivo, which are then infused
(re-infused) into a subject.
[0410] A subject may be any mammal, including non-human primate and
human subjects. Typically, a subject is a human subject.
[0411] In some embodiments, the bacterial vaccines are administered
to a subject (e.g., a mammalian subject, such as a human subject)
in an effective amount to induce an antigen-specific immune
response. The RNA encoding the bacterial antigen is expressed and
translated in vivo to produce the antigen, which then stimulates an
immune response in the subject.
[0412] Prophylactic protection from bacterial infections can be
achieved following administration of a bacterial RNA vaccine of the
present disclosure. Vaccines can be administered once, twice, three
times, four times or more but it is likely sufficient to administer
the vaccine once (optionally followed by a single booster). It is
possible, although less desirable, to administer the vaccine to an
infected individual to achieve a therapeutic response. Dosing may
need to be adjusted accordingly.
[0413] A method of eliciting an immune response in a subject
against a bacterial infection is provided in aspects of the present
disclosure. The method involves administering to the subject a
bacterial RNA vaccine comprising at least one RNA (e.g., mRNA)
having an open reading frame encoding at least one antigen, thereby
inducing in the subject an immune response specific to the antigen,
wherein anti-antigen antibody titer in the subject is increased
following vaccination relative to anti-antigen antibody titer in a
subject vaccinated with a prophylactically effective dose of a
traditional vaccine against the bacterium. An "anti-antigen
antibody" is a serum antibody the binds specifically to the
antigen.
[0414] A prophylactically effective dose is an effective dose that
prevents infection with the virus at a clinically acceptable level.
In some embodiments, the effective dose is a dose listed in a
package insert for the vaccine. A traditional vaccine, as used
herein, refers to a vaccine other than the mRNA vaccines of the
present disclosure. For instance, a traditional vaccine includes,
but is not limited, to live microorganism vaccines, killed
microorganism vaccines, subunit vaccines, protein antigen vaccines,
DNA vaccines, virus like particle (VLP) vaccines, etc. In exemplary
embodiments, a traditional vaccine is a vaccine that has achieved
regulatory approval and/or is registered by a national drug
regulatory body, for example the Food and Drug Administration (FDA)
in the United States or the European Medicines Agency (EMA).
[0415] In some embodiments, bacterial RNA (e.g. mRNA) vaccines
compositions may be administered at dosage levels sufficient to
deliver 0.0001 mg/kg to 100 mg/kg, 0.001 mg/kg to 0.05 mg/kg, 0.005
mg/kg to 0.05 mg/kg, 0.001 mg/kg to 0.005 mg/kg, 0.05 mg/kg to 0.5
mg/kg, 0.01 mg/kg to 50 mg/kg, 0.1 mg/kg to 40 mg/kg, 0.5 mg/kg to
30 mg/kg, 0.01 mg/kg to 10 mg/kg, 0.1 mg/kg to 10 mg/kg, or 1 mg/kg
to 25 mg/kg, of subject body weight per day, one or more times a
day, per week, per month, etc. to obtain the desired therapeutic,
diagnostic, prophylactic, or imaging effect (see, e.g., the range
of unit doses described in International Publication No
WO2013078199, the contents of which are herein incorporated by
reference in their entirety). The desired dosage may be delivered
three times a day, two times a day, once a day, every other day,
every third day, every week, every two weeks, every three weeks,
every four weeks, every 2 months, every three months, every 6
months, etc. In some embodiments, the desired dosage may be
delivered using multiple administrations (e.g., two, three, four,
five, six, seven, eight, nine, ten, eleven, twelve, thirteen,
fourteen, or more administrations). When multiple administrations
are employed, split dosing regimens such as those described herein
may be used. In exemplary embodiments, bacterial RNA (e.g., mRNA)
vaccines compositions may be administered at dosage levels
sufficient to deliver 0.0005 mg/kg to 0.01 mg/kg, e.g., about
0.0005 mg/kg to about 0.0075 mg/kg, e.g., about 0.0005 mg/kg, about
0.001 mg/kg, about 0.002 mg/kg, about 0.003 mg/kg, about 0.004
mg/kg or about 0.005 mg/kg.
[0416] In some embodiments, bacterial RNA (e.g., mRNA) vaccine
compositions may be administered once or twice (or more) at dosage
levels sufficient to deliver 0.025 mg/kg to 0.250 mg/kg, 0.025
mg/kg to 0.500 mg/kg, 0.025 mg/kg to 0.750 mg/kg, or 0.025 mg/kg to
1.0 mg/kg.
[0417] In some embodiments, bacterial RNA (e.g., mRNA) vaccine
compositions may be administered twice (e.g., Day 0 and Day 7, Day
0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60,
Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and
Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0
and 9 months later, Day 0 and 12 months later, Day 0 and 18 months
later, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0
and 10 years later) at a total dose of or at dosage levels
sufficient to deliver a total dose of 0.0100 mg, 0.025 mg, 0.050
mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg,
0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375
mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg,
0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700
mg, 0.725 mg, 0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg,
0.875 mg, 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg. Higher
and lower dosages and frequency of administration are encompassed
by the present disclosure. For example, a bacterial RNA (e.g.,
mRNA) vaccine composition may be administered three or four
times.
[0418] In some embodiments, bacterial RNA (e.g., mRNA) vaccine
compositions may be administered twice (e.g., Day 0 and Day 7, Day
0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60,
Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and
Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0
and 9 months later, Day 0 and 12 months later, Day 0 and 18 months
later, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0
and 10 years later) at a total dose of or at dosage levels
sufficient to deliver a total dose of 0.010 mg, 0.025 mg, 0.100 mg
or 0.400 mg.
[0419] In some embodiments, the bacterial RNA (e.g., mRNA) vaccine
for use in a method of vaccinating a subject is administered to the
subject as a single dosage of between 10 .mu.g/kg and 400 .mu.g/kg
of the nucleic acid vaccine (in an effective amount to vaccinate
the subject). In some embodiments the RNA (e.g., mRNA) vaccine for
use in a method of vaccinating a subject is administered to the
subject as a single dosage of between 10 .mu.g and 400 .mu.g of the
nucleic acid vaccine (in an effective amount to vaccinate the
subject). In some embodiments, a bacterial RNA (e.g., mRNA) vaccine
for use in a method of vaccinating a subject is administered to the
subject as a single dosage of 25-1000 .mu.g (e.g., a single dosage
of mRNA encoding bacterial antigen). In some embodiments, a
bacterial RNA (e.g., mRNA) vaccine is administered to the subject
as a single dosage of 25, 50, 100, 150, 200, 250, 300, 350, 400,
450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000
.mu.g. For example, a bacterial RNA (e.g., mRNA) vaccine may be
administered to a subject as a single dose of 25-100, 25-500,
50-100, 50-500, 50-1000, 100-500, 100-1000, 250-500, 250-1000, or
500-1000 .mu.g. In some embodiments, a bacterial RNA (e.g., mRNA)
vaccine for use in a method of vaccinating a subject is
administered to the subject as two dosages, the combination of
which equals 25-1000 .mu.g of the bacterial RNA (e.g., mRNA)
vaccine.
[0420] A bacterial RNA (e.g. mRNA) vaccine pharmaceutical
composition described herein can be formulated into a dosage form
described herein, such as an intranasal, intratracheal, or
injectable (e.g., intravenous, intraocular, intravitreal,
intramuscular, intradermal, intracardiac, intraperitoneal, and
subcutaneous).
Bacterial RNA (e.g., mRNA) Vaccine Formulations and Methods of
Use
[0421] Some aspects of the present disclosure provide formulations
of the bacterial RNA (e.g., mRNA) vaccine, wherein the RNA (e.g.,
mRNA) vaccine is formulated in an effective amount to produce an
antigen specific immune response in a subject (e.g., production of
antibodies specific to a bacterial antigenic polypeptide). "An
effective amount" is a dose of an RNA (e.g., mRNA) vaccine
effective to produce an antigen-specific immune response. Also
provided herein are methods of inducing an antigen-specific immune
response in a subject.
[0422] In some embodiments, the antigen-specific immune response is
characterized by measuring an anti-Streptococcus,
anti-Staphylococcus, and/or other bacterial antigenic polypeptide
antibody titer produced in a subject administered a bacterial RNA
(e.g., mRNA) vaccine as provided herein. An antibody titer is a
measurement of the amount of antibodies within a subject, for
example, antibodies that are specific to a particular antigen
(e.g., an anti-Streptococcus, anti-Staphylococcus, and/or
anti-bacterial antigenic polypeptide) or epitope of an antigen.
Antibody titer is typically expressed as the inverse of the
greatest dilution that provides a positive result. Enzyme-linked
immunosorbent assay (ELISA) is a common assay for determining
antibody titers, for example.
[0423] In some embodiments, an antibody titer is used to assess
whether a subject has had an infection or to determine whether
immunizations are required. In some embodiments, an antibody titer
is used to determine the strength of an autoimmune response, to
determine whether a booster immunization is needed, to determine
whether a previous vaccine was effective, and to identify any
recent or prior infections. In accordance with the present
disclosure, an antibody titer may be used to determine the strength
of an immune response induced in a subject by the bacterial RNA
(e.g., mRNA) vaccine.
[0424] In some embodiments, an anti-antigenic polypeptide (e.g., an
anti-Streptococcus, anti-Staphylococcus, and/or anti-bacterial
antigenic polypeptide) antibody titer produced in a subject is
increased 1 log to 10 log following vaccination relative to
anti-antigen antibody titer in a subject vaccinated with a
prophylactically effective dose of a traditional vaccine against
the bacterium or an unvaccinated subject. In some embodiments, the
anti-antigen antibody titer in the subject is increased 1 log, 2
log, 3 log, 4 log, 5 log, or 10 log following vaccination relative
to anti-antigen antibody titer in a subject vaccinated with a
prophylactically effective dose of a traditional vaccine against
the bacterium or an unvaccinated subject.
[0425] In some embodiments, the anti-antigenic polypeptide (e.g.,
an anti-Streptococcus, anti-Staphylococcus, and/or anti-bacterial
antigenic polypeptide) antibody titer produced in a subject is
increased at least 2 times relative to a control. For example, the
anti-antigenic polypeptide antibody titer produced in a subject may
be increased at least 3 times, at least 4 times, at least 5 times,
at least 6 times, at least 7 times, at least 8 times, at least 9
times, or at least 10 times relative to a control. In some
embodiments, the anti-antigenic polypeptide antibody titer produced
in the subject is increased 2, 3, 4, 5, 6, 7, 8, 9, or 10 times
relative to a control. In some embodiments, the anti-antigenic
polypeptide antibody titer produced in a subject is increased 2-10
times relative to a control. For example, the anti-antigenic
polypeptide antibody titer produced in a subject may be increased
2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6,
3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6,
6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10 times
relative to a control.
[0426] A control, in some embodiments, is the anti-antigenic
polypeptide (e.g., an anti-Streptococcus, anti-Staphylococcus,
and/or anti-bacterial antigenic polypeptide) antibody titer
produced in a subject who has not been administered a bacterial RNA
(e.g., mRNA) vaccine of the present disclosure. In some
embodiments, a control is an anti-antigenic polypeptide antibody
titer produced in a subject who has been administered a live
attenuated bacterial vaccine. An attenuated vaccine is a vaccine
produced by reducing the virulence of a viable (live). An
attenuated bacteria is altered in a manner that renders it harmless
or less virulent relative to live, unmodified bacteria. In some
embodiments, a control is an anti-antigenic polypeptide antibody
titer produced in a subject administered inactivated Streptococcus,
Staphylococcus, and/or other bacterial vaccine. In some
embodiments, a control is an anti-antigenic polypeptide antibody
titer produced in a subject administered a recombinant or purified
bacterial protein vaccine. Recombinant protein vaccines typically
include protein antigens that either have been produced in a
heterologous expression system (e.g., bacteria or yeast) or
purified from large amounts of the pathogenic organism. In some
embodiments, a control is an anti-antigenic polypeptide antibody
titer produced in a subject who has been administered a
Streptococcus, Staphylococcus, and/or other bacterial vaccine.
[0427] A method of eliciting an immune response in a subject
against a bacterium is provided in other aspects of the disclosure.
The method involves administering to the subject a bacterial RNA
vaccine comprising at least one RNA polynucleotide having an open
reading frame encoding at least one bacterial antigen, thereby
inducing in the subject an immune response specific to the
bacterial antigen, wherein the immune response in the subject is
equivalent to an immune response in a subject vaccinated with a
traditional vaccine against the bacterial antigen at 2 times to 100
times the dosage level relative to the RNA vaccine.
[0428] In some embodiments, the immune response in the subject is
equivalent to an immune response in a subject vaccinated with a
traditional vaccine at twice the dosage level relative to the
bacterial RNA vaccine. In some embodiments, the immune response in
the subject is equivalent to an immune response in a subject
vaccinated with a traditional vaccine at three times the dosage
level relative to the bacterial RNA vaccine. In some embodiments,
the immune response in the subject is equivalent to an immune
response in a subject vaccinated with a traditional vaccine at 4
times, 5 times, 10 times, 50 times, or 100 times the dosage level
relative to the bacterial RNA vaccine. In some embodiments, the
immune response in the subject is equivalent to an immune response
in a subject vaccinated with a traditional vaccine at 10 times to
1000 times the dosage level relative to the bacterial RNA vaccine.
In some embodiments, the immune response in the subject is
equivalent to an immune response in a subject vaccinated with a
traditional vaccine at 100 times to 1000 times the dosage level
relative to the bacterial RNA vaccine.
[0429] In other embodiments, the immune response is assessed by
determining [protein] antibody titer in the subject. In other
embodiments, the ability of serum or antibody from an immunized
subject is tested for its ability to neutralize viral uptake or
reduce the transformation of human B lymphocytes. In other
embodiments, the ability to promote a robust T cell response(s) is
measured using art recognized techniques.
[0430] Other aspects the disclosure provide methods of eliciting an
immune response in a subject against a bacterium by administering
to the subject a bacterial RNA vaccine comprising at least one RNA
polynucleotide having an open reading frame encoding at least one
bacterial antigen, thereby inducing in the subject an immune
response specific to the bacterial antigen, wherein the immune
response in the subject is induced 2 days to 10 weeks earlier
relative to an immune response induced in a subject vaccinated with
a prophylactically effective dose of a traditional vaccine against
the bacterium. In some embodiments, the immune response in the
subject is induced in a subject vaccinated with a prophylactically
effective dose of a traditional vaccine at 2 times to 100 times the
dosage level relative to the RNA vaccine.
[0431] In some embodiments, the immune response in the subject is
induced 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 5 weeks, or 10
weeks earlier relative to an immune response induced in a subject
vaccinated with a prophylactically effective dose of a traditional
vaccine.
[0432] Also provided herein are methods of eliciting an immune
response in a subject against a bacterium by administering to the
subject a bacterial RNA vaccine having an open reading frame
encoding a first antigen, wherein the RNA polynucleotide does not
include a stabilization element, and wherein an adjuvant is not
co-formulated or co-administered with the vaccine.
[0433] Bacterial RNA (e.g., mRNA) vaccines may be administered by
any route which results in a therapeutically effective outcome.
These include, but are not limited, to intradermal, intramuscular,
intranasal, and/or subcutaneous administration. The present
disclosure provides methods comprising administering RNA vaccines
to a subject in need thereof. The exact amount required will vary
from subject to subject, depending on the species, age, and general
condition of the subject, the severity of the disease, the
particular composition, its mode of administration, its mode of
activity, and the like. Bacterial RNA (e.g., mRNA) vaccines
compositions are typically formulated in dosage unit form for ease
of administration and uniformity of dosage. It will be understood,
however, that the total daily usage of bacterial RNA (e.g., mRNA)
vaccines compositions may be decided by the attending physician
within the scope of sound medical judgment. The specific
therapeutically effective, prophylactically effective, or
appropriate imaging dose level for any particular patient will
depend upon a variety of factors including the disorder being
treated and the severity of the disorder; the activity of the
specific compound employed; the specific composition employed; the
age, body weight, general health, sex and diet of the patient; the
time of administration, route of administration, and rate of
excretion of the specific compound employed; the duration of the
treatment; drugs used in combination or coincidental with the
specific compound employed; and like factors well known in the
medical arts.
[0434] The effective amount of a bacterial vaccine, as provided
herein, may be as low as 20 .mu.g, administered for example as a
single dose or as two 10 .mu.g doses. In some embodiments, the
effective amount is a total dose of 20 .mu.g-200 .mu.g. For
example, the effective amount may be a total dose of 20 .mu.g, 25
.mu.g, 30 .mu.g, 35 .mu.g, 40 .mu.g, 45 .mu.g, 50 .mu.g, 55 .mu.g,
60 .mu.g, 65 .mu.g, 70 .mu.g, 75 .mu.g, 80 .mu.g, 85 .mu.g, 90
.mu.g, 95 .mu.g, 100 .mu.g, 110 .mu.g, 120 .mu.g, 130 .mu.g, 140
.mu.g, 150 .mu.g, 160 .mu.g, 170 .mu.g, 180 .mu.g, 190 .mu.g or 200
.mu.g. In some embodiments, the effective amount is a total dose of
25 .mu.g-200 .mu.g. In some embodiments, the effective amount is a
total dose of 50 .mu.g-200 .mu.g.
[0435] In some embodiments, bacterial RNA (e.g., mRNA) vaccines
compositions may be administered at dosage levels sufficient to
deliver 0.0001 mg/kg to 100 mg/kg, 0.001 mg/kg to 0.05 mg/kg, 0.005
mg/kg to 0.05 mg/kg, 0.001 mg/kg to 0.005 mg/kg, 0.05 mg/kg to 0.5
mg/kg, 0.01 mg/kg to 50 mg/kg, 0.1 mg/kg to 40 mg/kg, 0.5 mg/kg to
30 mg/kg, 0.01 mg/kg to 10 mg/kg, 0.1 mg/kg to 10 mg/kg, or 1 mg/kg
to 25 mg/kg, of subject body weight per day, one or more times a
day, per week, per month, etc. to obtain the desired therapeutic,
diagnostic, prophylactic, or imaging effect (see e.g., the range of
unit doses described in International Publication No. WO2013078199,
herein incorporated by reference in its entirety). The desired
dosage may be delivered three times a day, two times a day, once a
day, every other day, every third day, every week, every two weeks,
every three weeks, every four weeks, every 2 months, every three
months, every 6 months, etc. In certain embodiments, the desired
dosage may be delivered using multiple administrations (e.g., two,
three, four, five, six, seven, eight, nine, ten, eleven, twelve,
thirteen, fourteen, or more administrations). When multiple
administrations are employed, split dosing regimens such as those
described herein may be used. In exemplary embodiments, bacterial
RNA (e.g., mRNA) vaccines compositions may be administered at
dosage levels sufficient to deliver 0.0005 mg/kg to 0.01 mg/kg,
e.g., about 0.0005 mg/kg to about 0.0075 mg/kg, e.g., about 0.0005
mg/kg, about 0.001 mg/kg, about 0.002 mg/kg, about 0.003 mg/kg,
about 0.004 mg/kg or about 0.005 mg/kg.
[0436] In some embodiments, bacterial RNA (e.g., mRNA) vaccine
compositions may be administered once or twice (or more) at dosage
levels sufficient to deliver 0.025 mg/kg to 0.250 mg/kg, 0.025
mg/kg to 0.500 mg/kg, 0.025 mg/kg to 0.750 mg/kg, or 0.025 mg/kg to
1.0 mg/kg.
[0437] In some embodiments, bacterial RNA (e.g., mRNA) vaccine
compositions may be administered twice (e.g., Day 0 and Day 7, Day
0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60,
Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and
Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0
and 9 months later, Day 0 and 12 months later, Day 0 and 18 months
later, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0
and 10 years later) at a total dose of or at dosage levels
sufficient to deliver a total dose of 0.0100 mg, 0.025 mg, 0.050
mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg,
0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375
mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg,
0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700
mg, 0.725 mg, 0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg,
0.875 mg, 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg. Higher
and lower dosages and frequency of administration are encompassed
by the present disclosure. For example, a bacterial RNA (e.g.,
mRNA) vaccine composition may be administered three or four
times.
[0438] In some embodiments, bacterial RNA (e.g., mRNA) vaccine
compositions may be administered twice (e.g., Day 0 and Day 7, Day
0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day 60,
Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 and
Day 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0
and 9 months later, Day 0 and 12 months later, Day 0 and 18 months
later, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0
and 10 years later) at a total dose of or at dosage levels
sufficient to deliver a total dose of 0.010 mg, 0.025 mg, 0.100 mg
or 0.400 mg.
[0439] In some embodiments, the bacterial RNA (e.g., mRNA) vaccine
for use in a method of vaccinating a subject is administered the
subject a single dosage of between 10 .mu.g/kg and 400 .mu.g/kg of
the nucleic acid vaccine in an effective amount to vaccinate the
subject. In some embodiments, the RNA vaccine for use in a method
of vaccinating a subject is administered the subject a single
dosage of between 10 .mu.g and 400 .mu.g of the nucleic acid
vaccine in an effective amount to vaccinate the subject. In some
embodiments, a bacterial RNA (e.g., mRNA) vaccine for use in a
method of vaccinating a subject is administered to the subject as a
single dosage of 25-1000 .mu.g (e.g., a single dosage of mRNA
encoding a bacterial antigen). In some embodiments, a bacterial RNA
vaccine is administered to the subject as a single dosage of 25,
50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,
700, 750, 800, 850, 900, 950 or 1000 .mu.g. For example, a
bacterial RNA vaccine may be administered to a subject as a single
dose of 25-100, 25-500, 50-100, 50-500, 50-1000, 100-500, 100-1000,
250-500, 250-1000, or 500-1000 .mu.g. In some embodiments, a
bacterial RNA (e.g., mRNA) vaccine for use in a method of
vaccinating a subject is administered to the subject as two
dosages, the combination of which equals 25-1000 .mu.g of the
bacterial RNA (e.g., mRNA) vaccine.
[0440] A bacterial RNA (e.g., mRNA) vaccine pharmaceutical
composition described herein can be formulated into a dosage form
described herein, such as an intranasal, intratracheal, or
injectable (e.g., intravenous, intraocular, intravitreal,
intramuscular, intradermal, intracardiac, intraperitoneal, and
subcutaneous).
Vaccine Efficacy
[0441] Some aspects of the present disclosure provide formulations
of the bacterial RNA (e.g., mRNA) vaccine, wherein the bacterial
RNA vaccine is formulated in an effective amount to produce an
antigen specific immune response in a subject (e.g., production of
antibodies specific to an antibacterial antigen). "An effective
amount" is a dose of a bacterial RNA (e.g., mRNA) vaccine effective
to produce an antigen-specific immune response. Also provided
herein are methods of inducing an antigen-specific immune response
in a subject.
[0442] As used herein, an immune response to a vaccine or LNP of
the present invention is the development in a subject of a humoral
and/or a cellular immune response to a (one or more) bacterial
protein(s) present in the vaccine. For purposes of the present
invention, a "humoral" immune response refers to an immune response
mediated by antibody molecules, including, e.g., secretory (IgA) or
IgG molecules, while a "cellular" immune response is one mediated
by T-lymphocytes (e.g., CD4+ helper and/or CD8+ T cells (e.g.,
CTLs) and/or other white blood cells. One important aspect of
cellular immunity involves an antigen-specific response by
cytolytic T-cells (CTLs). CTLs have specificity for peptide
antigens that are presented in association with proteins encoded by
the major histocompatibility complex (MHC) and expressed on the
surfaces of cells. CTLs help induce and promote the destruction of
intracellular microbes or the lysis of cells infected with such
microbes. Another aspect of cellular immunity involves and
antigen-specific response by helper T-cells. Helper T-cells act to
help stimulate the function, and focus the activity nonspecific
effector cells against cells displaying peptide antigens in
association with MHC molecules on their surface. A cellular immune
response also leads to the production of cytokines, chemokines, and
other such molecules produced by activated T-cells and/or other
white blood cells including those derived from CD4+ and CD8+
T-cells.
[0443] In some embodiments, the antigen-specific immune response is
characterized by measuring an antibacterial antigen antibody titer
produced in a subject administered a bacterial RNA (e.g., mRNA)
vaccine as provided herein. An antibody titer is a measurement of
the amount of antibodies within a subject, for example, antibodies
that are specific to a particular antigen (e.g., an antibacterial
antigen) or epitope of an antigen. Antibody titer is typically
expressed as the inverse of the greatest dilution that provides a
positive result. Enzyme-linked immunosorbent assay (ELISA) is a
common assay for determining antibody titers, for example.
[0444] In some embodiments, an antibody titer is used to assess
whether a subject has had an infection or to determine whether
immunizations are required. In some embodiments, an antibody titer
is used to determine the strength of an autoimmune response, to
determine whether a booster immunization is needed, to determine
whether a previous vaccine was effective, and to identify any
recent or prior infections. In accordance with the present
disclosure, an antibody titer may be used to determine the strength
of an immune response induced in a subject by the bacterial RNA
(e.g., mRNA) vaccine.
[0445] In some embodiments, an antibacterial antigen antibody titer
produced in a subject is increased by at least 1 log relative to a
control. For example, antibacterial antigen antibody titer produced
in a subject may be increased by at least 1.5, at least 2, at least
2.5, or at least 3 log relative to a control. In some embodiments,
the antibacterial antigen antibody titer produced in the subject is
increased by 1, 1.5, 2, 2.5 or 3 log relative to a control. In some
embodiments, the antibacterial antigen antibody titer produced in
the subject is increased by 1-3 log relative to a control. For
example, the antibacterial antigen antibody titer produced in a
subject may be increased by 1-1.5, 1-2, 1-2.5, 1-3, 1.5-2, 1.5-2.5,
1.5-3, 2-2.5, 2-3, or 2.5-3 log relative to a control.
[0446] In some embodiments, the antibacterial antigen antibody
titer produced in a subject is increased at least 2 times relative
to a control. For example, the antibacterial antigen antibody titer
produced in a subject may be increased at least 3 times, at least 4
times, at least 5 times, at least 6 times, at least 7 times, at
least 8 times, at least 9 times, or at least 10 times relative to a
control. In some embodiments, the antibacterial antigen antibody
titer produced in the subject is increased 2, 3, 4, 5, 6, 7, 8, 9,
or 10 times relative to a control. In some embodiments, the
antibacterial antigen antibody titer produced in a subject is
increased 2-10 times relative to a control. For example, the
antibacterial antigen antibody titer produced in a subject may be
increased 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8,
3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8,
5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9, or 9-10
times relative to a control.
[0447] A control, in some embodiments, is the antibacterial antigen
antibody titer produced in a subject who has not been administered
a bacterial RNA (e.g., mRNA) vaccine. In some embodiments, a
control is an antibacterial antigen antibody titer produced in a
subject administered a recombinant or purified bacterial protein
vaccine. Recombinant protein vaccines typically include protein
antigens that either have been produced in a heterologous
expression system (e.g., bacteria or yeast) or purified from large
amounts of the pathogenic organism.
[0448] In some embodiments, the ability of a bacterial vaccine to
be effective is measured in a murine model. For example, the
bacterial vaccines may be administered to a murine model and the
murine model assayed for induction of neutralizing antibody titers.
Viral challenge studies may also be used to assess the efficacy of
a vaccine of the present disclosure. For example, the bacterial
vaccines may be administered to a murine model, the murine model
challenged with a bacterium, and the murine model assayed for
survival and/or immune response (e.g., neutralizing antibody
response, T cell response (e.g., cytokine response)).
[0449] In some embodiments, an effective amount of a bacterial RNA
(e.g., mRNA) vaccine is a dose that is reduced compared to the
standard of care dose of a recombinant bacterial protein vaccine. A
"standard of care," as provided herein, refers to a medical or
psychological treatment guideline and can be general or specific.
"Standard of care" specifies appropriate treatment based on
scientific evidence and collaboration between medical professionals
involved in the treatment of a given condition. It is the
diagnostic and treatment process that a physician/clinician should
follow for a certain type of patient, illness or clinical
circumstance. A "standard of care dose," as provided herein, refers
to the dose of a recombinant or purified bacterial protein vaccine,
or a live attenuated or inactivated bacterial vaccine, or a
bacterial VLP vaccine, that a physician/clinician or other medical
professional would administer to a subject to treat or prevent a
bacterial infectoin, or a bacteria-related condition, while
following the standard of care guideline for treating or preventing
a bacterial infection, or a bacteria-related condition.
[0450] In some embodiments, the antibacterial antigen antibody
titer produced in a subject administered an effective amount of a
bacterial RNA vaccine is equivalent to an antibacterial antigen
antibody titer produced in a control subject administered a
standard of care dose of a recombinant or purified bacterial
protein vaccine, or a live attenuated or inactivated bacterial
vaccine, or a bacterial VLP vaccine.
[0451] In some embodiments, an effective amount of a bacterial RNA
(e.g., mRNA) vaccine is a dose equivalent to an at least 2-fold
reduction in a standard of care dose of a recombinant or purified
bacterial protein vaccine. For example, an effective amount of a
bacterial RNA vaccine may be a dose equivalent to an at least
3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least
7-fold, at least 8-fold, at least 9-fold, or at least 10-fold
reduction in a standard of care dose of a recombinant or purified
bacterial protein vaccine. In some embodiments, an effective amount
of a bacterial RNA vaccine is a dose equivalent to an at least at
least 100-fold, at least 500-fold, or at least 1000-fold reduction
in a standard of care dose of a recombinant or purified bacterial
protein vaccine. In some embodiments, an effective amount of a
bacterial RNA vaccine is a dose equivalent to a 2-, 3-, 4-, 5-, 6-,
7-, 8-, 9-, 10-, 20-, 50-, 100-, 250-, 500-, or 1000-fold reduction
in a standard of care dose of a recombinant or purified bacterial
protein vaccine. In some embodiments, the antibacterial antigen
antibody titer produced in a subject administered an effective
amount of a bacterial RNA vaccine is equivalent to an antibacterial
antigen antibody titer produced in a control subject administered
the standard of care dose of a recombinant protein or bacterial
protein vaccine, or a live attenuated or inactivated bacterial
vaccine, or a bacterial VLP vaccine. In some embodiments, an
effective amount of a bacterial RNA (e.g., mRNA) vaccine is a dose
equivalent to a 2-fold to 1000-fold (e.g., 2-fold to 100-fold,
10-fold to 1000-fold) reduction in the standard of care dose of a
recombinant or purified bacterial protein vaccine, wherein the
antibacterial antigen antibody titer produced in the subject is
equivalent to an antibacterial antigen antibody titer produced in a
control subject administered the standard of care dose of a
recombinant or purified bacterial protein vaccine, or a live
attenuated or inactivated bacterial vaccine, or a bacterial VLP
vaccine.
[0452] In some embodiments, the effective amount of a bacterial RNA
(e.g., mRNA) vaccine is a dose equivalent to a 2 to 1000-, 2 to
900-, 2 to 800-, 2 to 700-, 2 to 600-, 2 to 500-, 2 to 400-, 2 to
300-, 2 to 200-, 2 to 100-, 2 to 90-, 2 to 80-, 2 to 70-, 2 to 60-,
2 to 50-, 2 to 40-, 2 to 30-, 2 to 20-, 2 to 10-, 2 to 9-, 2 to 8-,
2 to 7-, 2 to 6-, 2 to 5-, 2 to 4-, 2 to 3-, 3 to 1000-, 3 to 900-,
3 to 800-, 3 to 700-, 3 to 600-, 3 to 500-, 3 to 400-, 3 to 3 to
00-, 3 to 200-, 3 to 100-, 3 to 90-, 3 to 80-, 3 to 70-, 3 to 60-,
3 to 50-, 3 to 40-, 3 to 30-, 3 to 20-, 3 to 10-, 3 to 9-, 3 to 8-,
3 to 7-, 3 to 6-, 3 to 5-, 3 to 4-, 4 to 1000-, 4 to 900-, 4 to
800-, 4 to 700-, 4 to 600-, 4 to 500-, 4 to 400-, 4 to 4 to 00-, 4
to 200-, 4 to 100-, 4 to 90-, 4 to 80-, 4 to 70-, 4 to 60-, 4 to
50, 4 to 40-, 4 to 30-, 4 to 20-, 4 to 10-, 4 to 9-, 4 to 8-, 4 to
7-, 4 to 6-, 4 to 5-, 4 to 4-, 5 to 1000-, 5 to 900-, 5 to 800-, 5
to 700-, 5 to 600-, 5 to 500-, 5 to 400-, 5 to 300-, 5 to 200-, 5
to 100-, 5 to 90-, 5 to 80-, 5 to 70-, 5 to 60-, 5 to 50-, 5 to
40-, 5 to 30-, 5 to 20-, 5 to 10-, 5 to 9-, 5 to 8, 5 to 7-, 5 to
6-, 6 to 1000-, 6 to 900-, 6 to 800-, 6 to 700-, 6 to 600-, 6 to
500-, 6 to 400-, 6 to 300-, 6 to 200-, 6 to 100-, 6 to 90-, 6 to
80-, 6 to 70-, 6 to 60-, 6 to 50-, 6 to 40-, 6 to 30-, 6 to 20-, 6
to 10-, 6 to 9-, 6 to 8-, 6 to 7-, 7 to 1000-, 7 to 900-, 7 to
800-, 7 to 700-, 7 to 600-, 7 to 500-, 7 to 400-, 7 to 300-, 7 to
200-, 7 to 100-, 7 to 90-, 7 to 80-, 7 to 70-, 7 to 60-, 7 to 50-,
7 to 40-, 7 to 30-, 7 to 20-, 7 to 10-, 7 to 9-, 7 to 8-, 8 to
1000-, 8 to 900-, 8 to 800-, 8 to 700-, 8 to 600-, 8 to 500-, 8 to
400-, 8 to 300-, 8 to 200-, 8 to 100-, 8 to 90-, 8 to 80-, 8 to
70-, 8 to 60-, 8 to 50-, 8 to 40-, 8 to 30-, 8 to 20-, 8 to 10-, 8
to 9-, 9 to 1000-, 9 to 900-, 9 to 800-, 9 to 700-, 9 to 600-, 9 to
500-, 9 to 400-, 9 to 300-, 9 to 200-, 9 to 100-, 9 to 90-, 9 to
80-, 9 to 70-, 9 to 60-, 9 to 50-, 9 to 40-, 9 to 30-, 9 to 20-, 9
to 10-, 10 to 1000-, 10 to 900-, 10 to 800-, 10 to 700-, 10 to
600-, 10 to 500-, 10 to 400-, 10 to 300-, 10 to 200-, 10 to 100-,
10 to 90-, 10 to 80-, 10 to 70-, 10 to 60-, 10 to 50-, 10 to 40-,
10 to 30-, 10 to 20-, 20 to 1000-, 20 to 900-, 20 to 800-, 20 to
700-, 20 to 600-, 20 to 500-, 20 to 400-, 20 to 300-, 20 to 200-,
20 to 100-, 20 to 90-, 20 to 80-, 20 to 70-, 20 to 60-, 20 to 50-,
20 to 40-, 20 to 30-, 30 to 1000-, 30 to 900-, 30 to 800-, 30 to
700-, 30 to 600-, 30 to 500-, 30 to 400-, 30 to 300-, 30 to 200-,
30 to 100-, 30 to 90-, 30 to 80-, 30 to 70-, 30 to 60-, 30 to 50-,
30 to 40-, 40 to 1000-, 40 to 900-, 40 to 800-, 40 to 700-, 40 to
600-, 40 to 500-, 40 to 400-, 40 to 300-, 40 to 200-, 40 to 100-,
40 to 90-, 40 to 80-, 40 to 70-, 40 to 60-, 40 to 50-, 50 to 1000-,
50 to 900-, 50 to 800-, 50 to 700-, 50 to 600-, 50 to 500-, 50 to
400-, 50 to 300-, 50 to 200-, 50 to 100-, 50 to 90-, 50 to 80-, 50
to 70-, 50 to 60-, 60 to 1000-, 60 to 900-, 60 to 800-, 60 to 700-,
60 to 600-, 60 to 500-, 60 to 400-, 60 to 300-, 60 to 200-, 60 to
100-, 60 to 90-, 60 to 80-, 60 to 70-, 70 to 1000-, 70 to 900-, 70
to 800-, 70 to 700-, 70 to 600-, 70 to 500-, 70 to 400-, 70 to
300-, 70 to 200-, 70 to 100-, 70 to 90-, 70 to 80-, 80 to 1000-, 80
to 900-, 80 to 800-, 80 to 700-, 80 to 600-, 80 to 500-, 80 to
400-, 80 to 300-, 80 to 200-, 80 to 100-, 80 to 90-, 90 to 1000-,
90 to 900-, 90 to 800-, 90 to 700-, 90 to 600-, 90 to 500-, 90 to
400-, 90 to 300-, 90 to 200-, 90 to 100-, 100 to 1000-, 100 to
900-, 100 to 800-, 100 to 700-, 100 to 600-, 100 to 500-, 100 to
400-, 100 to 300-, 100 to 200-, 200 to 1000-, 200 to 900-, 200 to
800-, 200 to 700-, 200 to 600-, 200 to 500-, 200 to 400-, 200 to
300-, 300 to 1000-, 300 to 900-, 300 to 800-, 300 to 700-, 300 to
600-, 300 to 500-, 300 to 400-, 400 to 1000-, 400 to 900-, 400 to
800-, 400 to 700-, 400 to 600-, 400 to 500-, 500 to 1000-, 500 to
900-, 500 to 800-, 500 to 700-, 500 to 600-, 600 to 1000-, 600 to
900-, 600 to 800-, 600 to 700-, 700 to 1000-, 700 to 900-, 700 to
800-, 800 to 1000-, 800 to 900-, or 900 to 1000-fold reduction in
the standard of care dose of a recombinant bacterial protein
vaccine. In some embodiments, such as the foregoing, the
antibacterial antigen antibody titer produced in the subject is
equivalent to an anti bacterial antigen antibody titer produced in
a control subject administered the standard of care dose of a
recombinant or purified bacterial protein vaccine, or a live
attenuated or inactivated bacterial vaccine, or a bacterial VLP
vaccine. In some embodiments, the effective amount is a dose
equivalent to (or equivalent to an at least) 2-, 3-, 4-, 5-, 6-,
7-, 8-, 9-, 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, 100-,
110-, 120-, 130-, 140-, 150-, 160-, 170-, 1280-, 190-, 200-, 210-,
220-, 230-, 240-, 250-, 260-, 270-, 280-, 290-, 300-, 310-, 320-,
330-, 340-, 350-, 360-, 370-, 380-, 390-, 400-, 410-, 420-, 430-,
440-, 450-, 4360-, 470-, 480-, 490-, 500-, 510-, 520-, 530-, 540-,
550-, 560-, 5760-, 580-, 590-, 600-, 610-, 620-, 630-, 640-, 650-,
660-, 670-, 680-, 690-, 700-, 710-, 720-, 730-, 740-, 750-, 760-,
770-, 780-, 790-, 800-, 810-, 820--, 830-, 840-, 850-, 860-, 870-,
880-, 890-, 900-, 910-, 920-, 930-, 940-, 950-, 960-, 970-, 980-,
990-, or 1000-fold reduction in the standard of care dose of a
recombinant bacterial protein vaccine. In some embodiments, such as
the foregoing, an antibacterial antigen antibody titer produced in
the subject is equivalent to an antibacterial antigen antibody
titer produced in a control subject administered the standard of
care dose of a recombinant or purified bacterial protein vaccine,
or a live attenuated or inactivated bacterial vaccine, or a
bacterial VLP vaccine.
[0453] In some embodiments, the effective amount of a bacterial RNA
(e.g., mRNA) vaccine is a total dose of 50-1000 .mu.g. In some
embodiments, the effective amount of a bacterial RNA (e.g., mRNA)
vaccine is a total dose of 50-1000, 50-900, 50-800, 50-700, 50-600,
50-500, 50-400, 50-300, 50-200, 50-100, 50-90, 50-80, 50-70, 50-60,
60-1000, 60-900, 60-800, 60-700, 60-600, 60-500, 60-400, 60-300,
60-200, 60-100, 60-90, 60-80, 60-70, 70-1000, 70-900, 70-800,
70-700, 70-600, 70-500, 70-400, 70-300, 70-200, 70-100, 70-90,
70-80, 80-1000, 80-900, 80-800, 80-700, 80-600, 80-500, 80-400,
80-300, 80-200, 80-100, 80-90, 90-1000, 90-900, 90-800, 90-700,
90-600, 90-500, 90-400, 90-300, 90-200, 90-100, 100-1000, 100-900,
100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200,
200-1000, 200-900, 200-800, 200-700, 200-600, 200-500, 200-400,
200-300, 300-1000, 300-900, 300-800, 300-700, 300-600, 300-500,
300-400, 400-1000, 400-900, 400-800, 400-700, 400-600, 400-500,
500-1000, 500-900, 500-800, 500-700, 500-600, 600-1000, 600-900,
600-900, 600-700, 700-1000, 700-900, 700-800, 800-1000, 800-900, or
900-1000 .mu.g. In some embodiments, the effective amount of a
bacterial RNA (e.g., mRNA) vaccine is a total dose of 50, 100, 150,
200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,
850, 900, 950 or 1000 .mu.g. In some embodiments, the effective
amount is a dose of 25-500 .mu.g administered to the subject a
total of two times. In some embodiments, the effective amount of a
bacterial RNA (e.g., mRNA) vaccine is a dose of 25-500, 25-400,
25-300, 25-200, 25-100, 25-50, 50-500, 50-400, 50-300, 50-200,
50-100, 100-500, 100-400, 100-300, 100-200, 150-500, 150-400,
150-300, 150-200, 200-500, 200-400, 200-300, 250-500, 250-400,
250-300, 300-500, 300-400, 350-500, 350-400, 400-500 or 450-500
.mu.g administered to the subject a total of two times. In some
embodiments, the effective amount of a bacterial RNA (e.g., mRNA)
vaccine is a total dose of 25, 50, 100, 150, 200, 250, 300, 350,
400, 450, or 500 .mu.g administered to the subject a total of two
times.
[0454] Vaccine efficacy may be assessed using standard analyses
(see, e.g., Weinberg et al., J Infect Dis. 2010 Jun. 1;
201(11):1607-10). For example, vaccine efficacy may be measured by
double-blind, randomized, clinical controlled trials. Vaccine
efficacy may be expressed as a proportionate reduction in disease
attack rate (AR) between the unvaccinated (ARU) and vaccinated
(ARV) study cohorts and can be calculated from the relative risk
(RR) of disease among the vaccinated group with use of the
following formulas:
Efficacy=(ARU-ARV)/ARU.times.100; and
Efficacy=(1-RR).times.100.
[0455] Likewise, vaccine effectiveness may be assessed using
standard analyses (see, e.g., Weinberg et al., J Infect Dis. 2010
Jun. 1; 201(11):1607-10). Vaccine effectiveness is an assessment of
how a vaccine (which may have already proven to have high vaccine
efficacy) reduces disease in a population. This measure can assess
the net balance of benefits and adverse effects of a vaccination
program, not just the vaccine itself, under natural field
conditions rather than in a controlled clinical trial. Vaccine
effectiveness is proportional to vaccine efficacy (potency) but is
also affected by how well target groups in the population are
immunized, as well as by other non-vaccine-related factors that
influence the `real-world` outcomes of hospitalizations, ambulatory
visits, or costs. For example, a retrospective case control
analysis may be used, in which the rates of vaccination among a set
of infected cases and appropriate controls are compared. Vaccine
effectiveness may be expressed as a rate difference, with use of
the odds ratio (OR) for developing infection despite
vaccination:
Effectiveness=(1-OR).times.100.
[0456] In some embodiments, efficacy of the bacterial vaccine is at
least 60% relative to unvaccinated control subjects. For example,
efficacy of the bacterial vaccine may be at least 65%, at least
70%, at least 75%, at least 80%, at least 85%, at least 95%, at
least 98%, or 100% relative to unvaccinated control subjects.
[0457] Sterilizing Immunity.
[0458] Sterilizing immunity refers to a unique immune status that
prevents effective pathogen infection into the host. In some
embodiments, the effective amount of a bacterial vaccine of the
present disclosure is sufficient to provide sterilizing immunity in
the subject for at least 1 year. For example, the effective amount
of a bacterial vaccine of the present disclosure is sufficient to
provide sterilizing immunity in the subject for at least 2 years,
at least 3 years, at least 4 years, or at least 5 years. In some
embodiments, the effective amount of a bacterial vaccine of the
present disclosure is sufficient to provide sterilizing immunity in
the subject at an at least 5-fold lower dose relative to control.
For example, the effective amount may be sufficient to provide
sterilizing immunity in the subject at an at least 10-fold lower,
15-fold, or 20-fold lower dose relative to a control.
[0459] Detectable Antigen.
[0460] In some embodiments, the effective amount of a bacterial
vaccine of the present disclosure is sufficient to produce
detectable levels of bacterial antigen as measured in serum of the
subject at 1-72 hours post administration.
[0461] Titer.
[0462] An antibody titer is a measurement of the amount of
antibodies within a subject, for example, antibodies that are
specific to a particular antigen (e.g., an antibacterial antigen).
Antibody titer is typically expressed as the inverse of the
greatest dilution that provides a positive result. Enzyme-linked
immunosorbent assay (ELISA) is a common assay for determining
antibody titers, for example.
[0463] In some embodiments, the effective amount of a bacterial
vaccine of the present disclosure is sufficient to produce a
1,000-10,000 neutralizing antibody titer produced by neutralizing
antibody against the bacterial antigen as measured in serum of the
subject at 1-72 hours post administration. In some embodiments, the
effective amount is sufficient to produce a 1,000-5,000
neutralizing antibody titer produced by neutralizing antibody
against the bacterial antigen as measured in serum of the subject
at 1-72 hours post administration. In some embodiments, the
effective amount is sufficient to produce a 5,000-10,000
neutralizing antibody titer produced by neutralizing antibody
against the bacterial antigen as measured in serum of the subject
at 1-72 hours post administration.
[0464] In some embodiments, the neutralizing antibody titer is at
least 100 NT.sub.50. For example, the neutralizing antibody titer
may be at least 200, 300, 400, 500, 600, 700, 800, 900 or 1000
NT.sub.50. In some embodiments, the neutralizing antibody titer is
at least 10,000 NT.sub.50.
[0465] In some embodiments, the neutralizing antibody titer is at
least 100 neutralizing units per milliliter (NU/mL). For example,
the neutralizing antibody titer may be at least 200, 300, 400, 500,
600, 700, 800, 900 or 1000 NU/mL. In some embodiments, the
neutralizing antibody titer is at least 10,000 NU/mL.
[0466] In some embodiments, an antibacterial antigen antibody titer
produced in the subject is increased by at least 1 log relative to
a control. For example, an antibacterial antigen antibody titer
produced in the subject may be increased by at least 2, 3, 4, 5, 6,
7, 8, 9 or 10 log relative to a control.
[0467] In some embodiments, an antibacterial antigen antibody titer
produced in the subject is increased at least 2 times relative to a
control. For example, an antibacterial antigen antibody titer
produced in the subject is increased by at least 3, 4, 5, 6, 7, 8,
9 or 10 times relative to a control.
[0468] In some embodiments, a geometric mean, which is the nth root
of the product of n numbers, is generally used to describe
proportional growth. Geometric mean, in some embodiments, is used
to characterize antibody titer produced in a subject.
[0469] A control may be, for example, an unvaccinated subject, or a
subject administered a live attenuated bacterial vaccine, an
inactivated bacterial vaccine, or a protein subunit bacterial
vaccine.
EQUIVALENTS
[0470] All references, patents and patent applications disclosed
herein are incorporated by reference with respect to the subject
matter for which each is cited, which in some cases may encompass
the entirety of the document.
[0471] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one." It should
also be understood that, unless clearly indicated to the contrary,
in any methods claimed herein that include more than one step or
act, the order of the steps or acts of the method is not
necessarily limited to the order in which the steps or acts of the
method are recited.
[0472] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," "composed of," and
the like are to be understood to be open-ended, i.e., to mean
including but not limited to. Only the transitional phrases
"consisting of" and "consisting essentially of" shall be closed or
semi-closed transitional phrases, respectively, as set forth in the
United States Patent Office Manual of Patent Examining Procedures,
Section 2111.03.
[0473] The terms "about" and "substantially" preceding a numerical
value mean.+-.10% of the recited numerical value.
[0474] Where a range of values is provided, each value between the
upper and lower ends of the range are specifically contemplated and
described herein.
[0475] The entire contents of International Application Nos.
PCT/US2015/02740, PCT/US2016/043348, PCT/US2016/043332,
PCT/US2016/058327, PCT/US2016/058324, PCT/US2016/058314,
PCT/US2016/058310, PCT/US2016/058321, PCT/US2016/058297,
PCT/US2016/058319, and PCT/US2016/058314 are incorporated herein by
reference.
EXAMPLES
Example 1: Manufacture of Polynucleotides
[0476] According to the present disclosure, the manufacture of
polynucleotides and/or parts or regions thereof may be accomplished
utilizing the methods taught in International Publication
WO2014/152027, entitled "Manufacturing Methods for Production of
RNA Transcripts," the contents of which is incorporated herein by
reference in its entirety.
[0477] Purification methods may include those taught in
International Publication WO2014/152030 and International
Publication WO2014/152031, each of which is incorporated herein by
reference in its entirety.
[0478] Detection and characterization methods of the
polynucleotides may be performed as taught in International
Publication WO2014/144039, which is incorporated herein by
reference in its entirety.
[0479] Characterization of the polynucleotides of the disclosure
may be accomplished using polynucleotide mapping, reverse
transcriptase sequencing, charge distribution analysis, detection
of RNA impurities, or any combination of two or more of the
foregoing. "Characterizing" comprises determining the RNA
transcript sequence, determining the purity of the RNA transcript,
or determining the charge heterogeneity of the RNA transcript, for
example. Such methods are taught in, for example, International
Publication WO2014/144711 and International Publication
WO2014/144767, the content of each of which is incorporated herein
by reference in its entirety.
Example 2: Chimeric Polynucleotide Synthesis
[0480] According to the present disclosure, two regions or parts of
a chimeric polynucleotide may be joined or ligated using
triphosphate chemistry. A first region or part of 100 nucleotides
or less is chemically synthesized with a 5' monophosphate and
terminal 3'desOH or blocked OH, for example. If the region is
longer than 80 nucleotides, it may be synthesized as two strands
for ligation.
[0481] If the first region or part is synthesized as a
non-positionally modified region or part using in vitro
transcription (IVT), conversion the 5'monophosphate with subsequent
capping of the 3' terminus may follow.
[0482] Monophosphate protecting groups may be selected from any of
those known in the art.
[0483] The second region or part of the chimeric polynucleotide may
be synthesized using either chemical synthesis or IVT methods. IVT
methods may include an RNA polymerase that can utilize a primer
with a modified cap. Alternatively, a cap of up to 130 nucleotides
may be chemically synthesized and coupled to the IVT region or
part.
[0484] For ligation methods, ligation with DNA T4 ligase, followed
by treatment with DNase should readily avoid concatenation.
[0485] The entire chimeric polynucleotide need not be manufactured
with a phosphate-sugar backbone. If one of the regions or parts
encodes a polypeptide, then such region or part may comprise a
phosphate-sugar backbone.
[0486] Ligation is then performed using any known click chemistry,
orthoclick chemistry, solulink, or other bioconjugate chemistries
known to those in the art.
Synthetic Route
[0487] The chimeric polynucleotide may be made using a series of
starting segments. Such segments include:
[0488] (a) a capped and protected 5' segment comprising a normal
3'OH (SEG. 1)
[0489] (b) a 5' triphosphate segment, which may include the coding
region of a polypeptide and a normal 3'OH (SEG. 2)
[0490] (c) a 5' monophosphate segment for the 3' end of the
chimeric polynucleotide (e.g., the tail) comprising cordycepin or
no 3'OH (SEG. 3)
[0491] After synthesis (chemical or IVT), segment 3 (SEG. 3) may be
treated with cordycepin and then with pyrophosphatase to create the
5' monophosphate.
[0492] Segment 2 (SEG. 2) may then be ligated to SEG. 3 using RNA
ligase. The ligated polynucleotide is then purified and treated
with pyrophosphatase to cleave the diphosphate. The treated SEG.
2-SEG. 3 construct may then be purified and SEG. 1 is ligated to
the 5' terminus. A further purification step of the chimeric
polynucleotide may be performed.
[0493] Where the chimeric polynucleotide encodes a polypeptide, the
ligated or joined segments may be represented as: 5'UTR (SEG. 1),
open reading frame or ORF (SEG. 2) and 3'UTR+PolyA (SEG. 3).
[0494] The yields of each step may be as much as 90-95%.
Example 3: PCR for cDNA Production
[0495] PCR procedures for the preparation of cDNA may be performed
using 2.times.KAPA HIFI.TM. HotStart ReadyMix by Kapa Biosystems
(Woburn, Mass.). This system includes 2.times.KAPA ReadyMix 12.5
.mu.l; Forward Primer (10 .mu.M) 0.75 .mu.l; Reverse Primer (10
.mu.M) 0.75 .mu.l; Template cDNA 100 ng; and dH.sub.20 diluted to
25.0 .mu.l. The reaction conditions may be at 95.degree. C. for 5
min. The reaction may be performed for 25 cycles of 98.degree. C.
for 20 sec, then 58.degree. C. for 15 sec, then 72.degree. C. for
45 sec, then 72.degree. C. for 5 min, then 4.degree. C. to
termination.
[0496] The reaction may be cleaned up using Invitrogen's
PURELINK.TM. PCR Micro Kit (Carlsbad, Calif.) per manufacturer's
instructions (up to 5 .mu.g). Larger reactions may require a
cleanup using a product with a larger capacity. Following the
cleanup, the cDNA may be quantified using the NANODROP.TM. and
analyzed by agarose gel electrophoresis to confirm that the cDNA is
the expected size. The cDNA may then be submitted for sequencing
analysis before proceeding to the in vitro transcription
reaction.
Example 4: In Vitro Transcription (IVT)
[0497] The in vitro transcription reaction generates RNA
polynucleotides. Such polynucleotides may comprise a region or part
of the polynucleotides of the disclosure, including chemically
modified RNA (e.g., mRNA) polynucleotides. The chemically modified
RNA polynucleotides can be uniformly modified polynucleotides. The
in vitro transcription reaction utilizes a custom mix of nucleotide
triphosphates (NTPs). The NTPs may comprise chemically modified
NTPs, or a mix of natural and chemically modified NTPs, or natural
NTPs.
[0498] A typical in vitro transcription reaction includes the
following:
TABLE-US-00001 1) Template cDNA 1.0 .mu.g 2) 10x transcription
buffer 2.0 .mu.l (400 mM Tris-HCl pH 8.0, 190 mM MgCl.sub.2, 50 mM
DTT, 10 mM Spermidine) 3) Custom NTPs (25 mM each) 0.2 .mu.l 4)
RNase Inhibitor 20 U 5) T7 RNA polymerase 3000 U 6) dH.sub.20 up to
20.0 .mu.l. and 7) Incubation at 37.degree. C. for 3 hr-5 hrs.
[0499] The crude IVT mix may be stored at 4.degree. C. overnight
for cleanup the next day. 1 U of RNase-free DNase may then be used
to digest the original template. After 15 minutes of incubation at
37.degree. C., the mRNA may be purified using Ambion's
MEGACLEAR.TM. Kit (Austin, Tex.) following the manufacturer's
instructions. This kit can purify up to 500 .mu.g of RNA. Following
the cleanup, the RNA polynucleotide may be quantified using the
NanoDrop and analyzed by agarose gel electrophoresis to confirm the
RNA polynucleotide is the proper size and that no degradation of
the RNA has occurred.
Example 5: Enzymatic Capping
[0500] Capping of a RNA polynucleotide is performed as follows
where the mixture includes: IVT RNA 60 .mu.g-180 .mu.g and
dH.sub.20 up to 72 .mu.l. The mixture is incubated at 65.degree. C.
for 5 minutes to denature RNA, and then is transferred immediately
to ice.
[0501] The protocol then involves the mixing of 10.times. Capping
Buffer (0.5 M Tris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl.sub.2)
(10.0 .mu.l); 20 mM GTP (5.0 .mu.l); 20 mM S-Adenosyl Methionine
(2.5 .mu.l); RNase Inhibitor (100 U); 2'-O-Methyltransferase
(400U); Vaccinia capping enzyme (Guanylyl transferase) (40 U);
dH.sub.20 (Up to 28 .mu.l); and incubation at 37.degree. C. for 30
minutes for 60 .mu.g RNA or up to 2 hours for 180 .mu.g of RNA.
[0502] The RNA polynucleotide may then be purified using Ambion's
MEGACLEAR.TM. Kit (Austin, Tex.) following the manufacturer's
instructions. Following the cleanup, the RNA may be quantified
using the NANODROP.TM. (ThermoFisher, Waltham, Mass.) and analyzed
by agarose gel electrophoresis to confirm the RNA polynucleotide is
the proper size and that no degradation of the RNA has occurred.
The RNA polynucleotide product may also be sequenced by running a
reverse-transcription-PCR to generate the cDNA for sequencing.
Example 6: PolyA Tailing Reaction
[0503] Without a poly-T in the cDNA, a poly-A tailing reaction must
be performed before cleaning the final product. This is done by
mixing capped IVT RNA (100 .mu.l); RNase Inhibitor (20 U);
10.times. Tailing Buffer (0.5 M Tris-HCl (pH 8.0), 2.5 M NaCl, 100
mM MgCl.sub.2) (12.0 .mu.l); 20 mM ATP (6.0 .mu.l); Poly-A
Polymerase (20 U); dH.sub.20 up to 123.5 .mu.l and incubation at
37.degree. C. for 30 min. If the poly-A tail is already in the
transcript, then the tailing reaction may be skipped and proceed
directly to cleanup with Ambion's MEGACLEAR.TM. kit (Austin, Tex.)
(up to 500 .mu.g). Poly-A Polymerase may be a recombinant enzyme
expressed in yeast.
[0504] It should be understood that the processivity or integrity
of the polyA tailing reaction may not always result in an exact
size polyA tail. Hence, polyA tails of approximately between 40-200
nucleotides, e.g., about 40, 50, 60, 70, 80, 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,
109, 110, 150-165, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164
or 165 are within the scope of the present disclosure.
Example 7: Natural 5' Caps and 5' Cap Analogues
[0505] 5'-capping of polynucleotides may be completed concomitantly
during the in vitro-transcription reaction using the following
chemical RNA cap analogs to generate the 5'-guanosine cap structure
according to manufacturer protocols: 3'-O-Me-m7G(5')ppp(5') G [the
ARCA cap]; G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A;
m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). 5'-capping
of modified RNA may be completed post-transcriptionally using a
Vaccinia Virus Capping Enzyme to generate the "Cap 0" structure:
m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). Cap 1
structure may be generated using both Vaccinia Virus Capping Enzyme
and a 2'-O methyl-transferase to generate:
m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structure may be generated from
the Cap 1 structure followed by the 2'-O-methylation of the
5'-antepenultimate nucleotide using a 2'-O methyl-transferase. Cap
3 structure may be generated from the Cap 2 structure followed by
the 2'-O-methylation of the 5'-preantepenultimate nucleotide using
a 2'-O methyl-transferase. Enzymes are preferably derived from a
recombinant source.
[0506] When transfected into mammalian cells, the modified mRNAs
have a stability of between 12-18 hours or more than 18 hours,
e.g., 24, 36, 48, 60, 72 or greater than 72 hours.
Example 8: Capping Assays
Protein Expression Assay
[0507] Polynucleotides (e.g., mRNA) encoding a polypeptide,
containing any of the caps taught herein, can be transfected into
cells at equal concentrations. The amount of protein secreted into
the culture medium can be assayed by ELISA at 6, 12, 24 and/or 36
hours post-transfection. Synthetic polynucleotides that secrete
higher levels of protein into the medium correspond to a synthetic
polynucleotide with a higher translationally-competent cap
structure.
Purity Analysis Synthesis
[0508] RNA (e.g., mRNA) polynucleotides encoding a polypeptide,
containing any of the caps taught herein can be compared for purity
using denaturing Agarose-Urea gel electrophoresis or HPLC analysis.
RNA polynucleotides with a single, consolidated band by
electrophoresis correspond to the higher purity product compared to
polynucleotides with multiple bands or streaking bands. Chemically
modified RNA polynucleotides with a single HPLC peak also
correspond to a higher purity product. The capping reaction with a
higher efficiency provides a more pure polynucleotide
population.
Cytokine Analysis
[0509] RNA (e.g., mRNA) polynucleotides encoding a polypeptide,
containing any of the caps taught herein can be transfected into
cells at multiple concentrations. The amount of pro-inflammatory
cytokines, such as TNF-alpha and IFN-beta, secreted into the
culture medium can be assayed by ELISA at 6, 12, 24 and/or 36 hours
post-transfection. RNA polynucleotides resulting in the secretion
of higher levels of pro-inflammatory cytokines into the medium
correspond to a polynucleotides containing an immune-activating cap
structure.
Capping Reaction Efficiency
[0510] RNA (e.g., mRNA) polynucleotides encoding a polypeptide,
containing any of the caps taught herein can be analyzed for
capping reaction efficiency by LC-MS after nuclease treatment.
Nuclease treatment of capped polynucleotides yield a mixture of
free nucleotides and the capped 5'-5-triphosphate cap structure
detectable by LC-MS. The amount of capped product on the LC-MS
spectra can be expressed as a percent of total polynucleotide from
the reaction and correspond to capping reaction efficiency. The cap
structure with a higher capping reaction efficiency has a higher
amount of capped product by LC-MS.
Example 9: Agarose Gel Electrophoresis of Modified RNA or RT PCR
Products
[0511] Individual RNA polynucleotides (200-400 ng in a 20 .mu.l
volume) or reverse transcribed PCR products (200-400 ng) may be
loaded into a well on a non-denaturing 1.2% Agarose E-Gel
(Invitrogen, Carlsbad, Calif.) and run for 12-15 minutes, according
to the manufacturer protocol.
Example 10: Nanodrop Modified RNA Quantification and UV Spectral
Data
[0512] Chemically modified RNA polynucleotides in TE buffer (1
.mu.l) are used for Nanodrop UV absorbance readings to quantitate
the yield of each polynucleotide from a chemical synthesis or in
vitro transcription reaction.
Example 11: Formulation of Modified mRNA Using Lipidoids
[0513] RNA (e.g., mRNA) polynucleotides may be formulated for in
vitro experiments by mixing the polynucleotides with the lipidoid
at a set ratio prior to addition to cells. In vivo formulation may
require the addition of extra ingredients to facilitate circulation
throughout the body. To test the ability of these lipidoids to form
particles suitable for in vivo work, a standard formulation process
used for siRNA-lipidoid formulations may be used as a starting
point. After formation of the particle, polynucleotide is added and
allowed to integrate with the complex. The encapsulation efficiency
is determined using a standard dye exclusion assays.
Example 12: Immunization of C57Bl/6 with Different Streptococcus
pneumoniae Pneumolysoid mRNA
[0514] Six- to eight-week old female C57Bl/6 mice were used to
study different Streptococcus pneumoniae pneumolysoid mRNA
vaccines. The vaccines were created using pneumolysin and
pneumolysin variants, including N-glycosylated mutants (NGM). The
mice were immunized intramuscularly on day 0 and then given a
booster dose three weeks later, on day 21. Blood samples were
collected for serum three days prior to the first immunization, on
day 20, and on approximately day 40-41. The groups are described in
Table 5.
TABLE-US-00002 TABLE 5 Overview of Immunization Study Groups Dose
MC3/ Dosage Vol 1.sup.st 2.sup.nd mRNA G# Antigen Route n = (ug)
(ul) Dose Dose conc 1 PlyD1 IM 5 10 50 Day Day 0.2 mg/ml 0 21 2
PlyD1 IM 5 2 50 Day Day 0.04 mg/ml 0 21 3 PlyD1_ IM 5 10 50 Day Day
0.2 mg/ml NGM 0 21 4 PlyD1_ IM 5 2 50 Day Day 0.04 mg/ml NGM 0 21 5
L460D IM 5 10 50 Day Day 0.2 mg/ml 0 21 6 L460D IM 5 2 50 Day Day
0.04 mg/ml 0 21 7 L460D_ IM 5 10 50 Day Day 0.2 mg/ml NGM 0 21 8
L460D_ IM 5 2 50 Day Day 0.04 mg/ml NGM 0 21 9 D205R IM 5 10 50 Day
Day 0.2 mg/ml 0 21 10 D205R IM 5 2 50 Day Day 0.04 mg/ml 0 21 11
D205R_ IM 5 10 50 Day Day 0.2 mg/ml NGM 0 21 12 D205R_ IM 5 2 50
Day Day 0.04 mg/ml NGM 0 21 13 Tris- IM 5 n/a 50 Day Day Sucrose 0
21
[0515] The serum samples were screened for anti-pneumolysin
antibodies and penuemolysin neutralization. As shown in FIGS. 1A
and 1B, the anti-pneumolysin IgG titers were roughly comparable or
higher than the PlyD1 and L460D protein-based vaccines. Relative to
the control, pneumolysoid mRNA elicited high serum IgG titers at
both 21 days (the booster dose, FIG. 1A) and at 41 days (FIG. 1B).
Mutating the N-glycosylation sites was shown to have a slight
negative effect on the total IgG titers.
[0516] The serum samples were also used in an immune serum
pneumolysin neutralization assay. In the assay, wild type
pneumolysin and the serum samples were incubated together and then
applied to red blood cells. Hemolysis of the red blood cells
resulted in an OD of 540 nm, which was measured and quantified. The
hemolytic unit (HU) determination (the pneumolysin concentration
for 50% hemolysis) is given in FIG. 2. The HU was found to be 5
ng/mL of pneumolysin. FIG. 3 shows the results of the pneumolysin
neutralization assay in the 10 .mu.g dose group at 41 days. The
pneumolysin neutralization assay was repeated using fresh red blood
cells at the 2 .mu.g and the 10 .mu.g doses. The results are shown
in FIG. 6. Mutating the N-glycosylation sites was shown to have a
positive effect on neutralization activity.
Example 13: Expression of mRNAs Encoding Pneumolysin, Characterized
Pneumolysin Toxoids, and Novel Pneumolysin Toxoids
[0517] The expression levels of mRNAs encoding pneumolysin,
characterized pneumolysin toxoids, and novel pneumolysin toxoid
variants in HEK293F cells were determined. Samples were run on gels
and stained with rabbit anti-His pAb (abcam ab9108) and mouse
anti-beta actin mAb. Previously characterized pneumolysin toxoid
variants are shown in FIG. 4 and novel pneumolysin toxoid variants
are shown in FIGS. 5A-5B.
[0518] The expression in vitro expression levels of different mRNA
constructs were measured as follows.
[0519] For pneumococcal surface protein A (PspA), which is
serovariable (there are more than 40 serotypes), and necessary for
virulence, constructs comprising one PspA from clades 1-2 and one
PspA from clades 3-5, preferably containing one that elicits the
most cross-protection, were used. The expression of S. pneumoniae
PspA truncation variant mRNAs from TIGR4 and Rx1 strains in HEK293
cells are shown in FIG. 7 and in Table 6 below. Samples were run on
gels and stained with rabbit anti-His antibody (A1647) and mouse
anti-actin mAb (Cy3).
TABLE-US-00003 TABLE 6 Summary of PspA Data Expected Obs. Size size
Obs. size Obs. Size Obs. Size (concentrated Name (kDa) (in vitro
QC) (cell lysate) (cell sup.) cell sup.)
Sp_PspA_noCBR_TIGR4_nlgK_cHis_no4A 57.854 78.468 ~70 ~70 ~70
Sp_PspA_noCBR_noPRR_TIGR4_nlgK_cHis_no4A 50.105 59.190 ~55 ~55 ~55
Sp_PspA_noCBR_Rx1_nlgK_cHis_no4A 50.418 69.799 ~62 ~62+ ~62-70
(faint) (glycosylated?) Sp_PspA_noCBR_noPRR_Rx1_nlgK_cHis_no4A
42.663 52.177 ~50 -- ~50-60 (glycosylated?)
[0520] Similar procedures were used to determine the expression
levels of constructs encoding choline-binding protein A (CbpA). The
Western blot was performed using PspA as a control. Samples were
run on gels and stained with rabbit anti-His antibody (A1647) and
mouse anti-actin mAb (Cy3) or with only rabbit anti-His antibody
(A1647). The results are shown in FIG. 8 and in Table 7 below.
[0521] Further, a liquid chromatograph-mass spectrometry (LC-MS/MS)
analysis was performed to detect specific protein expression in the
HEK293-F cell culture supernatant samples. Three samples (two
transfected, and one untransfected control) were used and analyzed
relative to two target proteins. Shotgun LC-MS/MS detected 46
peptides for both target proteins in the cell culture supernatants
(46 peptides from Cbpa_aa1_477; 31 of which were shared with
Cbpa_aa138-477).
TABLE-US-00004 TABLE 7 Summary of CbpA Data Concentration Expected
MW Observed MW mRNA Name (ng/ul) (kD) (kD)
Sp_Cbpa_aa1_477_nlgK_nHis 898.6667 56.740 80.571
Sp_Cbpa_aa138_477_nlgK_nHis 1322.667 41.573 62.335 AZ-eGFP-01-002
1233.566667
[0522] The expression levels of PhtD, PiuA, and PiaA were also
examined. The expression levels of each in HEK293 cells are shown
in FIG. 9 and in Table 8 below. Samples were run on gels and
stained with rabbit anti-His antibody (A1647) and mouse anti-actin
mAb (Cy3). All three were found to express the desired protein in
all three sample (lysate, supernatant, and concentrated
supernatant), indicating that both membrane-bound and secreted
proteins were recognized. As shown in Table 8, all observed
molecular weights (MW) were larger than the expected MW. Note that
the wild-type variant is usually slightly larger than the
N-glycosylation (NGM) mutant. Also, unspecific binding throughout
the lysate samples was observed at approximately 15 kD. In the
concentrated supernatant samples, unspecific binding was observed
at approximately 96 kD.
TABLE-US-00005 TABLE 8 Summary of PhtD, PiuA, and PiaA Data Final
Concentration Expected MW Observed MW mRNA Name (ng/ul) (kD) (kD)
Sp_PhtD_nlgK_cHis 612.60 94.762 ~99 Sp_PhtD_NGM_nlgK_cHis 608.59
94.592 ~97 Sp_PiuA_nlgk_cHis 784.08 34.546 ~45
Sp_PiuA_NGM_nlgk_cHis 948.34 34.484 ~38 Sp_PiaA_nlgk_cHis 1305.40
37.736 ~41 Sp_PiaA_NGM_nlgk_cHis 1113.39 37.69 ~41
[0523] Similarly, PcsB and StkP expression levels were examined
using anti-His and anti-actin staining (FIG. 10 and Table 9). PcsB
was found in the supernatant, but not the lysate, confirming that
it is a secreted antigen. In contrast StkP was found in the lysate,
but not the supernatant, indicating that it is a transmembrane
protein. Note that the wild type and N-glycosylation mutant (NGM)
appeared to be of the same size and intensity (FIG. 10). PcsB shows
some glycosylation, possibly at different sites.
TABLE-US-00006 TABLE 9 Summary of PcsB and StkP Data Expected
Observed Observed size size QC size Observed concentrated mRNA Name
(kDa) (kDa) size lysate supernatant Sp_PcsB_28_278_nlgK_cHis 29.876
33.629 -- ~50 Sp_PcsB_28_278_NGM_nlgK_cHis 29.800 33.167 -- ~40
Sp_StkP_345_659_nTMEM_cHis 36.826 42.532 ~45 --
Sp_StkP_345_659_NGM_nTMEM_cHis 36.750 42.721 ~45 --
[0524] Similar experiments were carried out for PsaA and PcpA. The
results are shown in FIG. 11 and in Table 10. Both appeared to be
mainly present in the concentrated supernatant samples.
TABLE-US-00007 TABLE 10 Summary of PsaA and PcpA Data Final
Concentration Expected MW QC MW Observed MW mRNA Name (ng/ul) (kD)
(kD) (kD) Sp_PsaA_nlgK_cHis 1138.289 35.471 40.315 ~39
Sp_PcpA_nlgK_cHis 1272.20653 51.541 56.044 ~56
Sp_PcpA_NGM_nlgK_cHis 1346.492 51.477 53.878 ~50 Az-eGFP-01-002
1233.570 -- -- --
[0525] The expression levels of SktP and PhtE were also examined
using similar methods. The results are shown in FIG. 12 and in
Table 11. StkP and the StkP N-glycosylated mutant were found to
express the desired protein in all three sample types (lysate,
supernatant, and concentrated supernatant). The concentrated
supernatant showed the strongest response. Both also had MWs that
were larger than expected. PhtE_nIgK was found to only express in
the concentrated supernatant sample with an accurate MW; however
its N-glycosylated mutant (PhtE_nIgK_NGM) was found to express an
accurate MW in all three samples. For both antigens, the
N-glycosylated mutant was found to be slightly smaller than the
wild type, and unspecific binding in the concentrated supernatant
samples was found to be present at around 96 kDa.
TABLE-US-00008 TABLE 11 Summary of SktP and PhtE Data Expected QC
Molecular Weight Measured MW Observed MW mRNA Name (kD) (kD) (kD)
Sp_StkP_364_659_noTMEM_cHis 34.76 29.45 ~43
Sp_StkP_364_659_noTMEM_NGM_cHis 34.68 35.65 ~42 Sp_PhtE_nlgK_cHis
115.21 138.72 ~115 Sp_PhtE_nlgK_NGM_cHis 115.04 136.509 ~112 GFP --
-- --
Example 14: Immunization of C57BL/6 with Different Streptococcus
pneumoniae mRNAs
[0526] Experiments similar to those described in Example 11 were
performed. Six- to eight-week old female C57Bl/6 mice were used to
study different Streptococcus pneumoniae pneumolysoid mRNA
vaccines. The vaccines were created encoding PspA fragments from
strains TIGR4 or Rx1, including N-glycosylated mutants (NGM). The
mice were immunized intramuscularly on day 0 and then given a
booster dose three weeks later, on day 21. Blood samples were
collected for serum three days prior to the first immunization, on
day 20, and on day 42. The groups are described in Table 12.
TABLE-US-00009 TABLE 12 Overview of Immunization Study Groups
(PspA) Dose MC3/ Dosage Vol 1.sup.st 2.sup.nd mRNA G# Antigen Route
n = (ug) (ul) Dose Dose conc 1 PspA_ IM 5 10 50 Day Day 0.2 mg/ml
noCBR_ 0 21 TIGR4 2 PspA_ IM 5 2 50 Day Day 0.04 mg/ml noCBR_ 0 21
TIGR4 3 PspA_ IM 5 10 50 Day Day 0.2 mg/ml noCBR_ 0 21 noPRR_ TIGR4
4 PspA_ IM 5 2 50 Day Day 0.04 mg/ml noCBR_ 0 21 noPRR_ TIGR4 5
PspA_ IM 5 10 50 Day Day 0.2 mg/ml noCBR_ 0 21 Rx1 6 PspA_ IM 5 2
50 Day Day 0.04 mg/ml noCBR_ 0 21 Rx1 7 PspA_ IM 5 10 50 Day Day
0.2 mg/ml noCBR_ 0 21 noPRR_ Rx1 8 PspA_ IM 5 2 50 Day Day 0.04
mg/ml noCBR_ 0 21 noPRR_ Rx1 9 PspA_ IM 5 10 50 Day Day 0.2 mg/ml
noCBR_ 0 21 Rx1 _ NGM 10 PspA_ IM 5 2 50 Day Day 0.04 mg/ml noCBR_
0 21 Rx1_ NGM 11 PspA_ IM 5 10 50 Day Day 0.2 mg/ml noCBR_ 0 21
noPRR_ Rx1_ NGM 12 PspA_ IM 5 2 50 Day Day 0.04 mg/ml noCBR_ 0 21
noPRR_ Rx1_ NGM 13 Tris- IM 5 n/a 50 Day Day Sucrose 0 21
[0527] Groups 5, 7, 9, and 11 were expected to have high titers.
The samples were screened for anti-PspA antibodies using an ELISA,
and the results are shown in FIG. 13. Few samples showed positive
titers, even with more concentrated serum samples.
[0528] The same study was repeated using CbpA variants. The groups
are described in Table 13 below.
TABLE-US-00010 TABLE 13 Overview of Immunization Study Groups
(CbpA) Dose MC3/ Dosage Vol 1.sup.st 2.sup.nd mRNA G# Antigen Route
n = (ug) (ul) Dose Dose conc 1 CbpA_ IM 5 10 50 Day Day 0.2 mg/ml
noCBR_ 0 21 TIGR4 2 CbpA_ IM 5 2 50 Day Day 0.04 mg/ml noCBR_ 0 21
TIGR4 3 CbpA_ IM 5 10 50 Day Day 0.2 mg/ml noCBR_ 0 21 noHVR_ TIGR4
4 CbpA_ IM 5 2 50 Day Day 0.04 mg/ml noCBR_ 0 21 noHVR_ TIGR4 5
Tris- IM 5 n/a 50 Day Day Sucrose 0 21
[0529] The ELISA results are shown in FIG. 14. The "no CBR" group
showed an increase in titer after the second dose: for the 10 .mu.g
dose, there was approximately a 11 og increase for the second dose;
and for the 2 .mu.g dose, there was an approximately 31 og
increase. The "noCBR_HVR" groups also showed an increase in titer
after the second dose: for the 10 .mu.g dose, there was
approximately a 21 og increase for the second dose; and for the 2
.mu.g dose, there was an approximately 11 og increase after the
second dose. However, there were two non-responders in the latter
group. Note that the 10 .mu.g dose was comparable for both
fragments, while the 2 .mu.g dose was much better for the no CBR
fragment.
[0530] The experiment was also carried out to test different
constructs encoding PiaA from the TIGR4 strain. The groups (n=5
mice per group) are shown in Table 14.
TABLE-US-00011 TABLE 14 Immunogenicity Study Groups (PiaA) Dose
MC3/ Dosage Vol 1.sup.st 2.sup.nd mRNA G# Antigen Route n = (ug)
(ul) Dose Dose conc 1 PiaA IM 5 10 50 Day Day 0.2 mg/ml 0 21 2 PiaA
IM 5 2 50 Day Day 0.04 mg/ml 0 21 3 PiaA_ IM 5 10 50 Day Day 0.2
mg/ml NGM 0 21 4 PiaA_ IM 5 2 50 Day Day 0.04 mg/ml NGM 0 21 5
Tris- IM 5 n/a 50 Day Day Sucrose 0 21
[0531] The ELISA results are shown in FIG. 15. A dose response was
seen in both the wild type and the N-glycosylated mutants assayed.
There was approximately a 21 og increase for all immunization
groups after the boost, which was similar among groups (Day 42).
However, the prime response was found to be more variable (Day 21).
Note that the wild type and N-glycosylation mutants appear to be
comparable.
[0532] The experiment was also performed to test different
constructs encoding PiuA from the TIGR4 strain. The groups (n=5
mice per group) are shown in Table 15.
TABLE-US-00012 TABLE 15 Immunogenicity Study Groups (PiuA) Dose
MC3/ Dosage Vol 1.sup.st 2.sup.nd mRNA G# Antigen Route n = (ug)
(ul) Dose Dose conc 1 PiuA IM 5 10 50 Day Day 0.2 mg/ml 0 21 2 PiuA
IM 5 2 50 Day Day 0.04 mg/ml 0 21 3 PiuA_ IM 5 10 50 Day Day 0.2
mg/ml NGM 0 21 4 PiuA_ IM 5 2 50 Day Day 0.04 mg/ml NGM 0 21 5
Tris- IM 5 n/a 50 Day Day Sucrose 0 21
[0533] The results are shown in FIG. 16. Like PiaA, PiuA is a
lipoprotein component of the iron ABC transporter and is highly
conserved. Together, they have found to be protective in a mouse
model of sepsis and pneumonia. As shown in FIG. 16, a dose response
was seen in the wild type PiuA constructs, while a reverse dose
response was observed in the N-glycosylated mutant constructs.
There was about a 11 og increase for all immunizations following
the boost, although the wild type constructs seemed to do slightly
better than the NGM versions. Note that initial prime titers were
high.
[0534] A similar experiment was performed using PhtD mRNA. Six- to
eight-week old female C57Bl/6 mice were used to study different
Streptococcus pneumoniae PhtD mRNA vaccines. The mice were
immunized intramuscularly on day 1 and then given a booster dose
three weeks later, on day 22. Blood samples were collected for
serum three days prior to the first immunization, on day 22, and on
day 36. On day 29, five mice per group were euthanized for T cell
analysis (via splenic samples). The groups are described in Table
16.
TABLE-US-00013 TABLE 16 Immunogenicity Study Groups (PhtD) Dose
MC3/ Dosage Vol 1.sup.st 2.sup.nd mRNA G# Antigen Route n = (ug)
(ul) Dose Dose conc 1 PhtD IM 10 10 50 Day Day 0.2 mg/ml 1 22 2
PhtD IM 10 2 50 Day Day 0.04 mg/ml 1 22 3 PhtD_ IM 10 10 50 Day Day
0.2 mg/ml NGM 1 22 4 PhtD_ IM 10 2 50 Day Day 0.04 mg/ml NGM 1 22 5
Tris- IM 10 n/a 50 Day Day Sucrose 1 22
[0535] Anti-PhtD antibody titers were measured in the serum samples
(FIG. 17). While a dose response was not observed, an increase of
at least 11 og was seen in the titers following the boost dose.
CD4+ T cell activation was determined from the splenic samples
(FIG. 18). While a slight CD8 IL-2 response was seen, its
percentage was very low. No response above the background level of
activation was observed in any other the screens.
Sequences
[0536] It should be understood that any of the mRNA sequences
described herein may include a 5' UTR and/or a 3' UTR. The UTR
sequences may be selected from the following sequences, or other
known UTR sequences may be used. It should also be understood that
any of the mRNA constructs described herein may further comprise a
polyA tail and/or cap (e.g., 7mG(5')ppp(5')NlmpNp). Further, while
many of the mRNAs and encoded antigen sequences described herein
include a signal peptide and/or a peptide tag (e.g., C-terminal His
tag), it should be understood that the indicated signal peptide
and/or peptide tag may be substituted for a different signal
peptide and/or peptide tag, or the signal peptide and/or peptide
tag may be omitted.
TABLE-US-00014 5' UTR: (SEQ ID NO: 54)
GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC 5' UTR: (SEQ ID NO:
69) CAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAA
UAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC 3' UTR: (SEQ ID NO: 55)
UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUC
CCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAA
UAAAGUCUGAGUGGGCGGC
TABLE-US-00015 TABLE 1 Pneumolysin Nucleic Acid Sequences
Description Sequence SEQ ID NO: S. pneumoniae pneumolysin gene,
complete cds; Accession No. M17717:
TTACAAGACCAACCTTGATTGACTTAGATAAGGTATTTATGTTGGATAATACGGTTATTCCGACTTCTTATCTA-
GCCAGACGGC
GACGCAATGTCTCAGAAGAATTGTACGAGGAAATTTTGGATCACTTAGTCCAACCACGGCTGATTTCGCTGAAC-
AAGTCTGAGT
TTATGCAACTCAATCCAGGAACTTATTAGGAGGAGAAGATGGCAAATAAAGCAGTAAATGACTTTATACTAGCT-
ATGAATTACG
ATAAAAAGAAACTCTTGACCCATCAGGGAGAAAGTATTGAAAATCGTTTCATCAAAGAGGGTAATCAGCTACCC-
GATGAGTTTG
TTGTTATCGAAAGAAAGAAGCGGAGCTTGTCGACAAATACAAGTGATATTTCTGTAACAGCTACCAACGACAGT-
CGCCTCTATC
CTGGAGCACTTCTCGTAGTGGATGAGACCTTGTTAGAGAATAATCCCACTCTTCTTGCGGTTGATCGTGCTCCG-
ATGACTTATA
GTATTGATTTGCCTGGTTTGGCAAGTAGCGATAGCTTTCTCCAAGTGGAAGACCCCAGCAATTCAAGTGTTCGC-
GGAGCGGTAA
ACGATTTGTTGGCTAAGTGGCATCAAGATTATGGTCAGGTCAATAATGTCCCAGCTAGAATGCAGTATGAAAAA-
ATAACGGCTC
ACAGCATGGAACAACTCAAGGTCAAGTTTGGTTCTGACTTTGAAAAGACAGGGAATTCTCTTGATATTGATTTT-
AACTCTGTCC
ATTCAGGTGAAAAGCAGATTCAGATTGTTAATTTTAAGCAGATTTATTATACAGTCAGCGTAGACGCTGTTAAA-
AATCCAGGAG
ATGTGTTTCAAGATACTGTAACGGTAGAGGATTTAAAACAGAGAGGAATTTCTGCAGAGCGTCCTTTGGTCTAT-
ATTTCGAGTG
TTGCTTATGGGCGCCAAGTCTATCTCAAGTTGGAAACCACGAGTAAGAGTGATGAAGTAGAGGCTGCTTTTGAA-
GCTTTGATAA
AAGGAGTCAAGGTAGCTCCTCAGACAGAGTGGAAGCAGATTTTGGACAATACAGAAGTGAAGGCGGTTATTTTA-
GGGGGCGACC
CAAGTTCGGGTGCCCGAGTTGTAACAGGCAAGGTGGATATGGTAGAGGACTTGATTCAAGAAGGCAGTCGCTTT-
ACAGCAGATC
ATCCAGGCTTGCCGATTTCCTATACAACTTCTTTTTTACGTGACAATGTAGTTGCGACCTTTCAAAACAGTACA-
GACTATGTTG
AGACTAAGGTTACAGCTTACAGAAACGGAGATTTACTGCTGGATCATAGTGGTGCCTATGTTGCCCAATATTAT-
ATTACTTGGG
ATGAATTATCCTATGATCATCAAGGTAAGGAAGTCTTGACTCCTAAGGCTTGGGACAGAAATGGGCAGGATTTG-
ACGGCTCACT
TTACCACTAGTATTCCTTTAAAAGGGAATGTTCGTAATCTCTCTGTCAAAATTAGAGAGTGTACCGGGCTTGCC-
TGGGAATGGT
GGCGTACGGTTTATGAAAAAACCGATTTGCCACTAGTGCGTAAGCGGACGATTTCTATTTGGGGAACAACTCTC-
TATCCTCAGG TAGAGGATAAGGTAGAAAATGACTAGGAGAGGAGAATGCTTGCGACAAAAAGA
(SEQ ID NO: 1) Sp_Ply_D205R_NGM_nIgK (5'UTR, ORF, 3' UTR)
TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGA-
AGAAATATAA
GAGCCACCATGGAAACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACCACCGGCATGGCC-
AACAAGGCCG
TGAACGACTTCATCCTGGCCATGAACTACGACAAGAAGAAGCTGCTGACCCACCAGGGCGAGAGCATCGAGAAC-
AGATTCATCA
AAGAGGGCAACCAGCTGCCCGACGAGTTCGTCGTGATCGAGCGGAAGAAGCGGAGCCTGAGCACCGACACCAGC-
GACATCAGCG
TGACCGCCACCAACGACGCCAGACTGTATCCTGGCGCTCTGCTGGTGGTGGACGAGACACTGCTGGAAAACAAC-
CCCATCCTGC
TGGCCGTGGACAGAGCCCCCATGACCTACAGCATCGACCTGCCTGGCCTGGCCAGCAGCGATAGCTTTCTGCAG-
GTGGAAGATC
CCAGCAACAGCGCCGTGCGGGGAGCCGTGAATGACCTGCTGGCTAAGTGGCACCAGGACTACGGCCAAGTGAAC-
AACGTGCCCG
CCAGAATGCAGTACGAGAAGATCACCGCCCACTCCATGGAACAGCTGAAAGTGAAGTTCGGCAGCGACTTCGAG-
AAAACCGGCA
ACAGCCTGGACATCGACTTCAACAGCGTGCACAGCGGCGAGAAGCAGATCCAGATCGTGAACTTCAAGCAGATC-
TACTACACCG
TGTCCGTGCGGGCCGTGAAGAACCCTGGGGACGTGTTCCAGGATACCGTGACCGTGGAAGATCTGAAGCAGCGG-
GGCATCAGCG
CCGAGAGGCCACTGGTGTACATCAGCAGCGTGGCCTACGGCAGACAGGTGTACCTGAAGCTGGAAACCACCTCC-
AAGAGCGACG
AGGTGGAAGCCGCCTTCGAGGCCCTGATCAAGGGCGTGAAAGTGGCCCCTCAGACCGAGTGGAAGCAGATTCTG-
GACAACACCG
AAGTGAAAGCCGTGATCCTGGGCGGCGACCCTTCTAGCGGAGCCAGAGTCGTGACAGGCAAGGTGGACATGGTG-
GAAGATCTGA
TCCAGGAAGGCAGCCGGTTCACCGCCGATCACCCTGGCCTGCCTATCAGCTACACCACAAGCTTTCTGAGAGAC-
AACGTGGTGG
CCACATTCCAGAACTCCGCCGACTACGTGGAAACAAAAGTGACCGCCTACCGGAACGGCGATCTGCTGCTGGAT-
CACTCCGGCG
CCTATGTGGCCCAGTACTACATCACCTGGGACGAGCTGAGCTACGATCACCAGGGCAAAGAGGTGCTGACCCCC-
AAGGCCTGGG
ACAGAAACGGCCAGGATCTGACAGCCCACTTCACAACCAGCATCCCCCTGAAGGGCAACGTGCGGAACCTGAGC-
GTGAAGATCA
GAGAGTGCACCGGACTGGCCTGGGAGTGGTGGCGGACCGTGTACGAAAAGACCGACCTGCCCCTCGTGCGGAAG-
CGGACCATCT
CTATCTGGGGCACCACGCTGTATCCTCAGGTGGAAGATAAGGTGGAAAACGACTGATAATAGGCTGGAGCCTCG-
GTGGCCATGC
TTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAG-
TCTGAGTGGG CGGC(SEQ ID NO: 2) SP_Ply_L460D_NGM_nIgK (5'UTR, ORF, 3'
UTR)
TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGA-
AGAAATATAA
GAGCCACCATGGAAACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACCACCGGCATGGCC-
AACAAGGCCG
TGAACGACTTCATCCTGGCCATGAACTACGACAAGAAGAAGCTGCTGACCCACCAGGGCGAGAGCATCGAGAAC-
AGATTCATCA
AAGAGGGCAACCAGCTGCCCGACGAGTTCGTCGTGATCGAGCGGAAGAAGCGGAGCCTGAGCACCGACACCAGC-
GACATCAGCG
TGACCGCCACCAACGACGCCAGACTGTATCCTGGCGCTCTGCTGGTGGTGGACGAGACACTGCTGGAAAACAAC-
CCCATCCTGC
TGGCCGTGGACAGAGCCCCCATGACCTACAGCATCGACCTGCCTGGCCTGGCCAGCAGCGATAGCTTTCTGCAG-
GTGGAAGATC
CCAGCAACAGCGCCGTGCGGGGAGCCGTGAATGACCTGCTGGCTAAGTGGCACCAGGACTACGGCCAAGTGAAC-
AACGTGCCCG
CCAGAATGCAGTACGAGAAGATCACCGCCCACTCCATGGAACAGCTGAAAGTGAAGTTCGGCAGCGACTTCGAG-
AAAACCGGCA
ACAGCCTGGACATCGACTTCAACAGCGTGCACAGCGGCGAGAAGCAGATCCAGATCGTGAACTTCAAGCAGATC-
TACTACACCG
TGTCCGTGGACGCCGTGAAGAACCCCGGGGACGTGTTCCAGGATACCGTGACCGTGGAAGATCTGAAGCAGCGG-
GGCATCAGCG
CCGAGAGGCCACTGGTGTACATCAGCAGCGTGGCCTACGGCAGACAGGTGTACCTGAAGCTGGAAACCACCTCC-
AAGAGCGACG
AGGTGGAAGCCGCCTTCGAGGCCCTGATCAAGGGCGTGAAAGTGGCCCCTCAGACCGAGTGGAAGCAGATTCTG-
GACAACACCG
AAGTGAAAGCCGTGATCCTGGGCGGCGACCCTTCTAGCGGAGCCAGAGTCGTGACAGGCAAGGTGGACATGGTG-
GAAGATCTGA
TCCAGGAAGGCAGCCGGTTCACCGCCGATCACCCTGGCCTGCCTATCAGCTACACCACAAGCTTTCTGAGAGAC-
AACGTGGTGG
CCACATTCCAGAACTCCGCCGACTACGTGGAAACAAAAGTGACCGCCTACCGGAACGGCGATCTGCTGCTGGAT-
CACTCCGGCG
CCTATGTGGCCCAGTACTACATCACCTGGGACGAGCTGAGCTACGATCACCAGGGCAAAGAGGTGCTGACCCCC-
AAGGCCTGGG
ACAGAAACGGCCAGGATCTGACAGCCCACTTCACAACCAGCATCCCCCTGAAGGGCAACGTGCGGAACCTGAGC-
GTGAAGATCA
GAGAGTGCACCGGACTGGCCTGGGAGTGGTGGCGGACCGTGTACGAAAAGACCGACCTGCCCCTCGTGCGGAAG-
CGGACCATCT
CTATCTGGGGCACCACCGACTACCCCCAGGTGGAAGATAAGGTGGAAAACGACTGATAATAGGCTGGAGCCTCG-
GTGGCCATGC
TTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAG-
TCTGAGTGGG CGGC(SEQ ID NO: 3) SP_Ply_T65C_G293C_C428A_PlyD1_
NGM_nIgK (5'UTR, ORF, 3' UTR)
TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGA-
AGAAATATAA
GAGCCACCATGGAAACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACCACCGGCATGGCC-
AACAAGGCCG
TGAACGACTTCATCCTGGCCATGAACTACGACAAGAAGAAGCTGCTGACCCACCAGGGCGAGAGCATCGAGAAC-
AGATTCATCA
AAGAGGGCAACCAGCTGCCCGACGAGTTCGTCGTGATCGAGCGGAAGAAGCGGAGCCTGAGCACCGACACCAGC-
GACATCAGCG
TGACCGCCTGCAACGACGCCAGACTGTATCCTGGCGCTCTGCTGGTGGTGGACGAGACACTGCTGGAAAACAAC-
CCCATCCTGC
TGGCCGTGGACAGAGCCCCCATGACCTACAGCATCGACCTGCCTGGCCTGGCCAGCAGCGATAGCTTTCTGCAG-
GTGGAAGATC
CCAGCAACAGCGCCGTGCGGGGAGCCGTGAATGACCTGCTGGCTAAGTGGCACCAGGACTACGGCCAAGTGAAC-
AACGTGCCCG
CCAGAATGCAGTACGAGAAGATCACCGCCCACTCCATGGAACAGCTGAAAGTGAAGTTCGGCAGCGACTTCGAG-
AAAACCGGCA
ACAGCCTGGACATCGACTTCAACAGCGTGCACAGCGGCGAGAAGCAGATCCAGATCGTGAACTTCAAGCAGATC-
TACTACACCG
TGTCCGTGGACGCCGTGAAGAACCCCGGGGACGTGTTCCAGGATACCGTGACCGTGGAAGATCTGAAGCAGCGG-
GGCATCAGCG
CCGAGAGGCCACTGGTGTACATCAGCAGCGTGGCCTACGGCAGACAGGTGTACCTGAAGCTGGAAACCACCTCC-
AAGAGCGACG
AGGTGGAAGCCGCCTTCGAGGCCCTGATCAAGGGCGTGAAAGTGGCCCCTCAGACCGAGTGGAAGCAGATTCTG-
GACAACACCG
AAGTGAAAGCCGTGATCCTGTGCGGCGACCCTTCTAGCGGAGCCAGAGTCGTGACAGGCAAGGTGGACATGGTG-
GAAGATCTGA
TCCAGGAAGGCAGCCGGTTCACCGCCGATCACCCTGGCCTGCCTATCAGCTACACCACAAGCTTTCTGAGAGAC-
AACGTGGTGG
CCACATTCCAGAACTCCGCCGACTACGTGGAAACAAAAGTGACAGCCTACCGGAACGGCGATCTGCTGCTGGAT-
CACTCCGGCG
CCTATGTGGCCCAGTACTACATCACCTGGGACGAGCTGAGCTACGATCACCAGGGCAAAGAGGTGCTGACCCCC-
AAGGCCTGGG
ACAGAAACGGCCAGGATCTGACAGCCCACTTCACAACCAGCATCCCCCTGAAGGGCAACGTGCGGAACCTGAGC-
GTGAAGATCA
GAGAAGCCACCGGACTGGCCTGGGAGTGGTGGCGGACAGTGTACGAAAAGACCGACCTGCCCCTCGTGCGGAAG-
CGGACCATCT
CTATCTGGGGCACCACGCTGTATCCTCAGGTGGAAGATAAGGTGGAAAACGACTGATAATAGGCTGGAGCCTCG-
GTGGCCATGC
TTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAG-
TCTGAGTGGG CGGC(SEQ ID NO: 4) SP_Ply_D205R_nIgK (5' UTR, ORF, 3'
UTR)
TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGA-
AGAAATATAA
GAGCCACCATGGAAACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACCACCGGCATGGCC-
AACAAGGCCG
TGAACGACTTCATCCTGGCCATGAACTACGACAAGAAGAAGCTGCTGACCCACCAGGGCGAGAGCATCGAGAAC-
AGATTCATCA
AAGAGGGCAACCAGCTGCCCGACGAGTTCGTCGTGATCGAGCGGAAGAAGCGGAGCCTGAGCACCAACACCAGC-
GACATCAGCG
TGACCGCCACCAACGACAGCAGACTGTATCCTGGCGCCCTGCTGGTGGTGGACGAGACACTGCTGGAAAACAAC-
CCCACCCTGC
TGGCCGTGGACAGAGCCCCTATGACCTACAGCATCGACCTGCCTGGCCTGGCCAGCAGCGATAGCTTTCTGCAG-
GTGGAAGATC
CCAGCAACAGCAGCGTGCGGGGAGCCGTGAATGACCTGCTGGCTAAGTGGCACCAGGACTACGGCCAAGTGAAC-
AACGTGCCCG
CCAGAATGCAGTACGAGAAGATCACCGCCCACTCCATGGAACAGCTGAAAGTGAAGTTCGGCAGCGACTTCGAG-
AAAACCGGCA
ACAGCCTGGACATCGACTTCAACAGCGTGCACAGCGGCGAGAAGCAGATCCAGATCGTGAACTTCAAGCAGATC-
TACTACACCG
TGTCCGTGCGGGCCGTGAAGAACCCTGGGGACGTGTTCCAGGATACCGTGACCGTGGAAGATCTGAAGCAGCGG-
GGCATCAGCG
CCGAGAGGCCACTGGTGTACATCAGCTCTGTGGCCTACGGCAGACAGGTGTACCTGAAGCTGGAAACCACCTCC-
AAGAGCGACG
AGGTGGAAGCCGCCTTCGAGGCCCTGATCAAGGGCGTGAAAGTGGCCCCTCAGACCGAGTGGAAGCAGATTCTG-
GACAACACCG
AAGTGAAAGCCGTGATCCTGGGCGGCGACCCTTCTAGCGGAGCCAGAGTCGTGACAGGCAAGGTGGACATGGTG-
GAAGATCTGA
TCCAGGAAGGCAGCCGGTTCACCGCCGATCACCCTGGCCTGCCTATCAGCTACACCACAAGCTTTCTGAGAGAC-
AACGTGGTGG
CCACATTCCAGAACAGCACCGACTACGTGGAAACAAAAGTGACCGCCTACCGGAACGGCGATCTGCTGCTGGAT-
CACTCCGGCG
CCTACGTGGCCCAGTACTACATCACCTGGGACGAGCTGAGCTACGATCACCAGGGCAAAGAGGTGCTGACCCCC-
AAGGCCTGGG
ACAGAAACGGCCAGGATCTGACAGCCCACTTCACAACCAGCATCCCCCTGAAGGGCAACGTGCGGAACCTGAGC-
GTGAAGATCA
GAGAGTGCACCGGACTGGCCTGGGAGTGGTGGCGGACCGTGTACGAAAAGACCGACCTGCCCCTCGTGCGGAAG-
CGGACCATCT
CTATCTGGGGCACCACGCTGTATCCTCAGGTGGAAGATAAGGTGGAAAACGACTGATAATAGGCTGGAGCCTCG-
GTGGCCATGC
TTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAG-
TCTGAGTGGG CGGC(SEQ ID NO: 30) SP_Ply_L460D_nIgK (5' UTR, ORF, 3'
UTR)
TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGA-
AGAAATATAA
GAGCCACCATGGAAACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACCACCGGCATGGCC-
AACAAGGCCG
TGAACGACTTCATCCTGGCCATGAACTACGACAAGAAGAAGCTGCTGACCCACCAGGGCGAGAGCATCGAGAAC-
AGATTCATCA
AAGAGGGCAACCAGCTGCCCGACGAGTTCGTCGTGATCGAGCGGAAGAAGCGGAGCCTGAGCACCAACACCAGC-
GACATCAGCG
TGACCGCCACCAACGACAGCAGACTGTATCCTGGCGCCCTGCTGGTGGTGGACGAGACACTGCTGGAAAACAAC-
CCCACCCTGC
TGGCCGTGGACAGAGCCCCTATGACCTACAGCATCGACCTGCCTGGCCTGGCCAGCAGCGATAGCTTTCTGCAG-
GTGGAAGATC
CCAGCAACAGCAGCGTGCGGGGAGCCGTGAATGACCTGCTGGCTAAGTGGCACCAGGACTACGGCCAAGTGAAC-
AACGTGCCCG
CCAGAATGCAGTACGAGAAGATCACCGCCCACTCCATGGAACAGCTGAAAGTGAAGTTCGGCAGCGACTTCGAG-
AAAACCGGCA
ACAGCCTGGACATCGACTTCAACAGCGTGCACAGCGGCGAGAAGCAGATCCAGATCGTGAACTTCAAGCAGATC-
TACTACACCG
TGTCCGTGGACGCCGTGAAGAACCCCGGGGACGTGTTCCAGGATACCGTGACCGTGGAAGATCTGAAGCAGCGG-
GGCATCAGCG
CCGAGAGGCCACTGGTGTACATCAGCTCTGTGGCCTACGGCAGACAGGTGTACCTGAAGCTGGAAACCACCTCC-
AAGAGCGACG
AGGTGGAAGCCGCCTTCGAGGCCCTGATCAAGGGCGTGAAAGTGGCCCCTCAGACCGAGTGGAAGCAGATTCTG-
GACAACACCG
AAGTGAAAGCCGTGATCCTGGGCGGCGACCCTTCTAGCGGAGCCAGAGTCGTGACAGGCAAGGTGGACATGGTG-
GAAGATCTGA
TCCAGGAAGGCAGCCGGTTCACCGCCGATCACCCTGGCCTGCCTATCAGCTACACCACAAGCTTTCTGAGAGAC-
AACGTGGTGG
CCACATTCCAGAACAGCACCGACTACGTGGAAACAAAAGTGACCGCCTACCGGAACGGCGATCTGCTGCTGGAT-
CACTCCGGCG
CCTACGTGGCCCAGTACTACATCACCTGGGACGAGCTGAGCTACGATCACCAGGGCAAAGAGGTGCTGACCCCC-
AAGGCCTGGG
ACAGAAACGGCCAGGATCTGACAGCCCACTTCACAACCAGCATCCCCCTGAAGGGCAACGTGCGGAACCTGAGC-
GTGAAGATCA
GAGAGTGCACCGGACTGGCCTGGGAGTGGTGGCGGACCGTGTACGAAAAGACCGACCTGCCCCTCGTGCGGAAG-
CGGACCATCT
CTATCTGGGGCACCACCGATTACCCCCAGGTGGAAGATAAGGTGGAAAACGACTGATAATAGGCTGGAGCCTCG-
GTGGCCATGC
TTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAG-
TCTGAGTGGG CGGC(SEQ ID NO: 31) SP_Ply_T65C_G293C_C428A_PlyD1_nIgK
(5' UTR, ORF, 3' UTR)
TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGA-
AGAAATATAA
GAGCCACCATGGAAACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACCACCGGCATGGCC-
AACAAGGCCG
TGAACGACTTCATCCTGGCCATGAACTACGACAAGAAGAAGCTGCTGACCCACCAGGGCGAGAGCATCGAGAAC-
AGATTCATCA
AAGAGGGCAACCAGCTGCCCGACGAGTTCGTCGTGATCGAGCGGAAGAAGCGGAGCCTGAGCACCAACACCAGC-
GACATCAGCG
TGACCGCCTGCAACGACAGCAGACTGTATCCTGGCGCCCTGCTGGTGGTGGACGAGACACTGCTGGAAAACAAC-
CCCACCCTGC
TGGCCGTGGACAGAGCCCCTATGACCTACAGCATCGACCTGCCTGGCCTGGCCAGCAGCGATAGCTTTCTGCAG-
GTGGAAGATC
CCAGCAACAGCAGCGTGCGGGGAGCCGTGAATGACCTGCTGGCTAAGTGGCACCAGGACTACGGCCAAGTGAAC-
AACGTGCCCG
CCAGAATGCAGTACGAGAAGATCACCGCCCACTCCATGGAACAGCTGAAAGTGAAGTTCGGCAGCGACTTCGAG-
AAAACCGGCA
ACAGCCTGGACATCGACTTCAACAGCGTGCACAGCGGCGAGAAGCAGATCCAGATCGTGAACTTCAAGCAGATC-
TACTACACCG
TGTCCGTGGACGCCGTGAAGAACCCCGGGGACGTGTTCCAGGATACCGTGACCGTGGAAGATCTGAAGCAGCGG-
GGCATCAGCG
CCGAGAGGCCACTGGTGTACATCAGCTCTGTGGCCTACGGCAGACAGGTGTACCTGAAGCTGGAAACCACCTCC-
AAGAGCGACG
AGGTGGAAGCCGCCTTCGAGGCCCTGATCAAGGGCGTGAAAGTGGCCCCTCAGACCGAGTGGAAGCAGATTCTG-
GACAACACCG
AAGTGAAAGCCGTGATCCTGTGCGGCGACCCTTCTAGCGGAGCCAGAGTCGTGACAGGCAAGGTGGACATGGTG-
GAAGATCTGA
TCCAGGAAGGCAGCCGGTTCACCGCCGATCACCCTGGCCTGCCTATCAGCTACACCACAAGCTTTCTGAGAGAC-
AACGTGGTGG
CCACATTCCAGAACAGCACCGACTACGTGGAAACAAAAGTGACAGCCTACCGGAACGGCGATCTGCTGCTGGAT-
CACTCCGGCG
CCTACGTGGCCCAGTACTACATCACCTGGGACGAGCTGAGCTACGATCACCAGGGCAAAGAGGTGCTGACCCCC-
AAGGCCTGGG
ACAGAAACGGCCAGGATCTGACAGCCCACTTCACAACCAGCATCCCCCTGAAGGGCAACGTGCGGAACCTGAGC-
GTGAAGATCA
GAGAAGCCACCGGACTGGCCTGGGAGTGGTGGCGGACAGTGTACGAAAAGACCGACCTGCCCCTCGTGCGGAAG-
CGGACCATCT
CTATCTGGGGCACCACGCTGTATCCTCAGGTGGAAGATAAGGTGGAAAACGACTGATAATAGGCTGGAGCCTCG-
GTGGCCATGC
TTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAG-
TCTGAGTGGG CGGC(SEQ ID NO: 32) Sp_Ply_D205R_NGM_nIgK (ORF)
ATGGAAACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACCACCGGCATGGCCAACAAGGC-
CGTGAACGAC
TTCATCCTGGCCATGAACTACGACAAGAAGAAGCTGCTGACCCACCAGGGCGAGAGCATCGAGAACAGATTCAT-
CAAAGAGGGC
AACCAGCTGCCCGACGAGTTCGTCGTGATCGAGCGGAAGAAGCGGAGCCTGAGCACCGACACCAGCGACATCAG-
CGTGACCGCC
ACCAACGACGCCAGACTGTATCCTGGCGCTCTGCTGGTGGTGGACGAGACACTGCTGGAAAACAACCCCATCCT-
GCTGGCCGTG
GACAGAGCCCCCATGACCTACAGCATCGACCTGCCTGGCCTGGCCAGCAGCGATAGCTTTCTGCAGGTGGAAGA-
TCCCAGCAAC
AGCGCCGTGCGGGGAGCCGTGAATGACCTGCTGGCTAAGTGGCACCAGGACTACGGCCAAGTGAACAACGTGCC-
CGCCAGAATG
CAGTACGAGAAGATCACCGCCCACTCCATGGAACAGCTGAAAGTGAAGTTCGGCAGCGACTTCGAGAAAACCGG-
CAACAGCCTG
GACATCGACTTCAACAGCGTGCACAGCGGCGAGAAGCAGATCCAGATCGTGAACTTCAAGCAGATCTACTACAC-
CGTGTCCGTG
CGGGCCGTGAAGAACCCTGGGGACGTGTTCCAGGATACCGTGACCGTGGAAGATCTGAAGCAGCGGGGCATCAG-
CGCCGAGAGG
CCACTGGTGTACATCAGCAGCGTGGCCTACGGCAGACAGGTGTACCTGAAGCTGGAAACCACCTCCAAGAGCGA-
CGAGGTGGAA
GCCGCCTTCGAGGCCCTGATCAAGGGCGTGAAAGTGGCCCCTCAGACCGAGTGGAAGCAGATTCTGGACAACAC-
CGAAGTGAAA
GCCGTGATCCTGGGCGGCGACCCTTCTAGCGGAGCCAGAGTCGTGACAGGCAAGGTGGACATGGTGGAAGATCT-
GATCCAGGAA
GGCAGCCGGTTCACCGCCGATCACCCTGGCCTGCCTATCAGCTACACCACAAGCTTTCTGAGAGACAACGTGGT-
GGCCACATTC
CAGAACTCCGCCGACTACGTGGAAACAAAAGTGACCGCCTACCGGAACGGCGATCTGCTGCTGGATCACTCCGG-
CGCCTATGTG
GCCCAGTACTACATCACCTGGGACGAGCTGAGCTACGATCACCAGGGCAAAGAGGTGCTGACCCCCAAGGCCTG-
GGACAGAAAC
GGCCAGGATCTGACAGCCCACTTCACAACCAGCATCCCCCTGAAGGGCAACGTGCGGAACCTGAGCGTGAAGAT-
CAGAGAGTGC
ACCGGACTGGCCTGGGAGTGGTGGCGGACCGTGTACGAAAAGACCGACCTGCCCCTCGTGCGGAAGCGGACCAT-
CTCTATCTGG GGCACCACGCTGTATCCTCAGGTGGAAGATAAGGTGGAAAACGAC(SEQ ID NO:
56) SP_Ply_L460D_NGM_nIgK (ORF)
ATGGAAACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACCACCGGCATGGCCAACAAGGC-
CGTGAACGAC
TTCATCCTGGCCATGAACTACGACAAGAAGAAGCTGCTGACCCACCAGGGCGAGAGCATCGAGAACAGATTCAT-
CAAAGAGGGC
AACCAGCTGCCCGACGAGTTCGTCGTGATCGAGCGGAAGAAGCGGAGCCTGAGCACCGACACCAGCGACATCAG-
CGTGACCGCC
ACCAACGACGCCAGACTGTATCCTGGCGCTCTGCTGGTGGTGGACGAGACACTGCTGGAAAACAACCCCATCCT-
GCTGGCCGTG
GACAGAGCCCCCATGACCTACAGCATCGACCTGCCTGGCCTGGCCAGCAGCGATAGCTTTCTGCAGGTGGAAGA-
TCCCAGCAAC
AGCGCCGTGCGGGGAGCCGTGAATGACCTGCTGGCTAAGTGGCACCAGGACTACGGCCAAGTGAACAACGTGCC-
CGCCAGAATG
CAGTACGAGAAGATCACCGCCCACTCCATGGAACAGCTGAAAGTGAAGTTCGGCAGCGACTTCGAGAAAACCGG-
CAACAGCCTG
GACATCGACTTCAACAGCGTGCACAGCGGCGAGAAGCAGATCCAGATCGTGAACTTCAAGCAGATCTACTACAC-
CGTGTCCGTG
GACGCCGTGAAGAACCCCGGGGACGTGTTCCAGGATACCGTGACCGTGGAAGATCTGAAGCAGCGGGGCATCAG-
CGCCGAGAGG
CCACTGGTGTACATCAGCAGCGTGGCCTACGGCAGACAGGTGTACCTGAAGCTGGAAACCACCTCCAAGAGCGA-
CGAGGTGGAA
GCCGCCTTCGAGGCCCTGATCAAGGGCGTGAAAGTGGCCCCTCAGACCGAGTGGAAGCAGATTCTGGACAACAC-
CGAAGTGAAA
GCCGTGATCCTGGGCGGCGACCCTTCTAGCGGAGCCAGAGTCGTGACAGGCAAGGTGGACATGGTGGAAGATCT-
GATCCAGGAA
GGCAGCCGGTTCACCGCCGATCACCCTGGCCTGCCTATCAGCTACACCACAAGCTTTCTGAGAGACAACGTGGT-
GGCCACATTC
CAGAACTCCGCCGACTACGTGGAAACAAAAGTGACCGCCTACCGGAACGGCGATCTGCTGCTGGATCACTCCGG-
CGCCTATGTG
GCCCAGTACTACATCACCTGGGACGAGCTGAGCTACGATCACCAGGGCAAAGAGGTGCTGACCCCCAAGGCCTG-
GGACAGAAAC
GGCCAGGATCTGACAGCCCACTTCACAACCAGCATCCCCCTGAAGGGCAACGTGCGGAACCTGAGCGTGAAGAT-
CAGAGAGTGC
ACCGGACTGGCCTGGGAGTGGTGGCGGACCGTGTACGAAAAGACCGACCTGCCCCTCGTGCGGAAGCGGACCAT-
CTCTATCTGG GGCACCACCGACTACCCCCAGGTGGAAGATAAGGTGGAAAACGAC(SEQ ID NO:
57) SP_Ply_T65C_G293C_C428A_PlyD1_ NGM_nIgK (ORF)
ATGGAAACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACCACCGGCATGGCCAACAAGGC-
CGTGAACGAC
TTCATCCTGGCCATGAACTACGACAAGAAGAAGCTGCTGACCCACCAGGGCGAGAGCATCGAGAACAGATTCAT-
CAAAGAGGGC
AACCAGCTGCCCGACGAGTTCGTCGTGATCGAGCGGAAGAAGCGGAGCCTGAGCACCGACACCAGCGACATCAG-
CGTGACCGCC
TGCAACGACGCCAGACTGTATCCTGGCGCTCTGCTGGTGGTGGACGAGACACTGCTGGAAAACAACCCCATCCT-
GCTGGCCGTG
GACAGAGCCCCCATGACCTACAGCATCGACCTGCCTGGCCTGGCCAGCAGCGATAGCTTTCTGCAGGTGGAAGA-
TCCCAGCAAC
AGCGCCGTGCGGGGAGCCGTGAATGACCTGCTGGCTAAGTGGCACCAGGACTACGGCCAAGTGAACAACGTGCC-
CGCCAGAATG
CAGTACGAGAAGATCACCGCCCACTCCATGGAACAGCTGAAAGTGAAGTTCGGCAGCGACTTCGAGAAAACCGG-
CAACAGCCTG
GACATCGACTTCAACAGCGTGCACAGCGGCGAGAAGCAGATCCAGATCGTGAACTTCAAGCAGATCTACTACAC-
CGTGTCCGTG
GACGCCGTGAAGAACCCCGGGGACGTGTTCCAGGATACCGTGACCGTGGAAGATCTGAAGCAGCGGGGCATCAG-
CGCCGAGAGG
CCACTGGTGTACATCAGCAGCGTGGCCTACGGCAGACAGGTGTACCTGAAGCTGGAAACCACCTCCAAGAGCGA-
CGAGGTGGAA
GCCGCCTTCGAGGCCCTGATCAAGGGCGTGAAAGTGGCCCCTCAGACCGAGTGGAAGCAGATTCTGGACAACAC-
CGAAGTGAAA
GCCGTGATCCTGTGCGGCGACCCTTCTAGCGGAGCCAGAGTCGTGACAGGCAAGGTGGACATGGTGGAAGATCT-
GATCCAGGAA
GGCAGCCGGTTCACCGCCGATCACCCTGGCCTGCCTATCAGCTACACCACAAGCTTTCTGAGAGACAACGTGGT-
GGCCACATTC
CAGAACTCCGCCGACTACGTGGAAACAAAAGTGACAGCCTACCGGAACGGCGATCTGCTGCTGGATCACTCCGG-
CGCCTATGTG
GCCCAGTACTACATCACCTGGGACGAGCTGAGCTACGATCACCAGGGCAAAGAGGTGCTGACCCCCAAGGCCTG-
GGACAGAAAC
GGCCAGGATCTGACAGCCCACTTCACAACCAGCATCCCCCTGAAGGGCAACGTGCGGAACCTGAGCGTGAAGAT-
CAGAGAAGCC
ACCGGACTGGCCTGGGAGTGGTGGCGGACAGTGTACGAAAAGACCGACCTGCCCCTCGTGCGGAAGCGGACCAT-
CTCTATCTGG GGCACCACGCTGTATCCTCAGGTGGAAGATAAGGTGGAAAACGAC(SEQ ID NO:
58) SP_Ply_D205R_nIgK (ORF)
ATGGAAACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACCACCGGCATGGCCAACAAGGC-
CGTGAACGAC
TTCATCCTGGCCATGAACTACGACAAGAAGAAGCTGCTGACCCACCAGGGCGAGAGCATCGAGAACAGATTCAT-
CAAAGAGGGC
AACCAGCTGCCCGACGAGTTCGTCGTGATCGAGCGGAAGAAGCGGAGCCTGAGCACCAACACCAGCGACATCAG-
CGTGACCGCC
ACCAACGACAGCAGACTGTATCCTGGCGCCCTGCTGGTGGTGGACGAGACACTGCTGGAAAACAACCCCACCCT-
GCTGGCCGTG
GACAGAGCCCCTATGACCTACAGCATCGACCTGCCTGGCCTGGCCAGCAGCGATAGCTTTCTGCAGGTGGAAGA-
TCCCAGCAAC
AGCAGCGTGCGGGGAGCCGTGAATGACCTGCTGGCTAAGTGGCACCAGGACTACGGCCAAGTGAACAACGTGCC-
CGCCAGAATG
CAGTACGAGAAGATCACCGCCCACTCCATGGAACAGCTGAAAGTGAAGTTCGGCAGCGACTTCGAGAAAACCGG-
CAACAGCCTG
GACATCGACTTCAACAGCGTGCACAGCGGCGAGAAGCAGATCCAGATCGTGAACTTCAAGCAGATCTACTACAC-
CGTGTCCGTG
CGGGCCGTGAAGAACCCTGGGGACGTGTTCCAGGATACCGTGACCGTGGAAGATCTGAAGCAGCGGGGCATCAG-
CGCCGAGAGG
CCACTGGTGTACATCAGCTCTGTGGCCTACGGCAGACAGGTGTACCTGAAGCTGGAAACCACCTCCAAGAGCGA-
CGAGGTGGAA
GCCGCCTTCGAGGCCCTGATCAAGGGCGTGAAAGTGGCCCCTCAGACCGAGTGGAAGCAGATTCTGGACAACAC-
CGAAGTGAAA
GCCGTGATCCTGGGCGGCGACCCTTCTAGCGGAGCCAGAGTCGTGACAGGCAAGGTGGACATGGTGGAAGATCT-
GATCCAGGAA
GGCAGCCGGTTCACCGCCGATCACCCTGGCCTGCCTATCAGCTACACCACAAGCTTTCTGAGAGACAACGTGGT-
GGCCACATTC
CAGAACAGCACCGACTACGTGGAAACAAAAGTGACCGCCTACCGGAACGGCGATCTGCTGCTGGATCACTCCGG-
CGCCTACGTG
GCCCAGTACTACATCACCTGGGACGAGCTGAGCTACGATCACCAGGGCAAAGAGGTGCTGACCCCCAAGGCCTG-
GGACAGAAAC
GGCCAGGATCTGACAGCCCACTTCACAACCAGCATCCCCCTGAAGGGCAACGTGCGGAACCTGAGCGTGAAGAT-
CAGAGAGTGC
ACCGGACTGGCCTGGGAGTGGTGGCGGACCGTGTACGAAAAGACCGACCTGCCCCTCGTGCGGAAGCGGACCAT-
CTCTATCTGG GGCACCACGCTGTATCCTCAGGTGGAAGATAAGGTGGAAAACGAC(SEQ ID NO:
59) SP_Ply_L460D_nigK (ORF)
ATGGAAACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACCACCGGCATGGCCAACAAGGC-
CGTGAACGAC
TTCATCCTGGCCATGAACTACGACAAGAAGAAGCTGCTGACCCACCAGGGCGAGAGCATCGAGAACAGATTCAT-
CAAAGAGGGC
AACCAGCTGCCCGACGAGTTCGTCGTGATCGAGCGGAAGAAGCGGAGCCTGAGCACCAACACCAGCGACATCAG-
CGTGACCGCC
ACCAACGACAGCAGACTGTATCCTGGCGCCCTGCTGGTGGTGGACGAGACACTGCTGGAAAACAACCCCACCCT-
GCTGGCCGTG
GACAGAGCCCCTATGACCTACAGCATCGACCTGCCTGGCCTGGCCAGCAGCGATAGCTTTCTGCAGGTGGAAGA-
TCCCAGCAAC
AGCAGCGTGCGGGGAGCCGTGAATGACCTGCTGGCTAAGTGGCACCAGGACTACGGCCAAGTGAACAACGTGCC-
CGCCAGAATG
CAGTACGAGAAGATCACCGCCCACTCCATGGAACAGCTGAAAGTGAAGTTCGGCAGCGACTTCGAGAAAACCGG-
CAACAGCCTG
GACATCGACTTCAACAGCGTGCACAGCGGCGAGAAGCAGATCCAGATCGTGAACTTCAAGCAGATCTACTACAC-
CGTGTCCGTG
GACGCCGTGAAGAACCCCGGGGACGTGTTCCAGGATACCGTGACCGTGGAAGATCTGAAGCAGCGGGGCATCAG-
CGCCGAGAGG
CCACTGGTGTACATCAGCTCTGTGGCCTACGGCAGACAGGTGTACCTGAAGCTGGAAACCACCTCCAAGAGCGA-
CGAGGTGGAA
GCCGCCTTCGAGGCCCTGATCAAGGGCGTGAAAGTGGCCCCTCAGACCGAGTGGAAGCAGATTCTGGACAACAC-
CGAAGTGAAA
GCCGTGATCCTGGGCGGCGACCCTTCTAGCGGAGCCAGAGTCGTGACAGGCAAGGTGGACATGGTGGAAGATCT-
GATCCAGGAA
GGCAGCCGGTTCACCGCCGATCACCCTGGCCTGCCTATCAGCTACACCACAAGCTTTCTGAGAGACAACGTGGT-
GGCCACATTC
CAGAACAGCACCGACTACGTGGAAACAAAAGTGACCGCCTACCGGAACGGCGATCTGCTGCTGGATCACTCCGG-
CGCCTACGTG
GCCCAGTACTACATCACCTGGGACGAGCTGAGCTACGATCACCAGGGCAAAGAGGTGCTGACCCCCAAGGCCTG-
GGACAGAAAC
GGCCAGGATCTGACAGCCCACTTCACAACCAGCATCCCCCTGAAGGGCAACGTGCGGAACCTGAGCGTGAAGAT-
CAGAGAGTGC
ACCGGACTGGCCTGGGAGTGGTGGCGGACCGTGTACGAAAAGACCGACCTGCCCCTCGTGCGGAAGCGGACCAT-
CTCTATCTGG GGCACCACCGATTACCCCCAGGTGGAAGATAAGGTGGAAAACGAC(SEQ ID NO:
60) SP_Ply_T65C_G293C_C428A_PlyD1_nIgK (ORF)
ATGGAAACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACCACCGGCATGGCCAACAAGGC-
CGTGAACGAC
TTCATCCTGGCCATGAACTACGACAAGAAGAAGCTGCTGACCCACCAGGGCGAGAGCATCGAGAACAGATTCAT-
CAAAGAGGGC
AACCAGCTGCCCGACGAGTTCGTCGTGATCGAGCGGAAGAAGCGGAGCCTGAGCACCAACACCAGCGACATCAG-
CGTGACCGCC
TGCAACGACAGCAGACTGTATCCTGGCGCCCTGCTGGTGGTGGACGAGACACTGCTGGAAAACAACCCCACCCT-
GCTGGCCGTG
GACAGAGCCCCTATGACCTACAGCATCGACCTGCCTGGCCTGGCCAGCAGCGATAGCTTTCTGCAGGTGGAAGA-
TCCCAGCAAC
AGCAGCGTGCGGGGAGCCGTGAATGACCTGCTGGCTAAGTGGCACCAGGACTACGGCCAAGTGAACAACGTGCC-
CGCCAGAATG
CAGTACGAGAAGATCACCGCCCACTCCATGGAACAGCTGAAAGTGAAGTTCGGCAGCGACTTCGAGAAAACCGG-
CAACAGCCTG
GACATCGACTTCAACAGCGTGCACAGCGGCGAGAAGCAGATCCAGATCGTGAACTTCAAGCAGATCTACTACAC-
CGTGTCCGTG
GACGCCGTGAAGAACCCCGGGGACGTGTTCCAGGATACCGTGACCGTGGAAGATCTGAAGCAGCGGGGCATCAG-
CGCCGAGAGG
CCACTGGTGTACATCAGCTCTGTGGCCTACGGCAGACAGGTGTACCTGAAGCTGGAAACCACCTCCAAGAGCGA-
CGAGGTGGAA
GCCGCCTTCGAGGCCCTGATCAAGGGCGTGAAAGTGGCCCCTCAGACCGAGTGGAAGCAGATTCTGGACAACAC-
CGAAGTGAAA
GCCGTGATCCTGTGCGGCGACCCTTCTAGCGGAGCCAGAGTCGTGACAGGCAAGGTGGACATGGTGGAAGATCT-
GATCCAGGAA
GGCAGCCGGTTCACCGCCGATCACCCTGGCCTGCCTATCAGCTACACCACAAGCTTTCTGAGAGACAACGTGGT-
GGCCACATTC
CAGAACAGCACCGACTACGTGGAAACAAAAGTGACAGCCTACCGGAACGGCGATCTGCTGCTGGATCACTCCGG-
CGCCTACGTG
GCCCAGTACTACATCACCTGGGACGAGCTGAGCTACGATCACCAGGGCAAAGAGGTGCTGACCCCCAAGGCCTG-
GGACAGAAAC
GGCCAGGATCTGACAGCCCACTTCACAACCAGCATCCCCCTGAAGGGCAACGTGCGGAACCTGAGCGTGAAGAT-
CAGAGAAGCC
ACCGGACTGGCCTGGGAGTGGTGGCGGACAGTGTACGAAAAGACCGACCTGCCCCTCGTGCGGAAGCGGACCAT-
CTCTATCTGG GGCACCACGCTGTATCCTCAGGTGGAAGATAAGGTGGAAAACGAC(SEQ ID NO:
61) mRNA Sequences S. pneumoniae pneumolysin gene, complete cds;
Accession No. M17717
UUACAAGACCAACCUUGAUUGACUUAGAUAAGGUAUUUAUGUUGGAUAAUACGGUUAUUCCGACUUCUUAUCUA-
GCCAGACGGC
GACGCAAUGUCUCAGAAGAAUUGUACGAGGAAAUUUUGGAUCACUUAGUCCAACCACGGCUGAUUUCGCUGAAC-
AAGUCUGAGU
UUAUGCAACUCAAUCCAGGAACUUAUUAGGAGGAGAAGAUGGCAAAUAAAGCAGUAAAUGACUUUAUACUAGCU-
AUGAAUUACG
AUAAAAAGAAACUCUUGACCCAUCAGGGAGAAAGUAUUGAAAAUCGUUUCAUCAAAGAGGGUAAUCAGCUACCC-
GAUGAGUUUG
UUGUUAUCGAAAGAAAGAAGCGGAGCUUGUCGACAAAUACAAGUGAUAUUUCUGUAACAGCUACCAACGACAGU-
CGCCUCUAUC
CUGGAGCACUUCUCGUAGUGGAUGAGACCUUGUUAGAGAAUAAUCCCACUCUUCUUGCGGUUGAUCGUGCUCCG-
AUGACUUAUA
GUAUUGAUUUGCCUGGUUUGGCAAGUAGCGAUAGCUUUCUCCAAGUGGAAGACCCCAGCAAUUCAAGUGUUCGC-
GGAGCGGUAA
ACGAUUUGUUGGCUAAGUGGCAUCAAGAUUAUGGUCAGGUCAAUAAUGUCCCAGCUAGAAUGCAGUAUGAAAAA-
AUAACGGCUC
ACAGCAUGGAACAACUCAAGGUCAAGUUUGGUUCUGACUUUGAAAAGACAGGGAAUUCUCUUGAUAUUGAUUUU-
AACUCUGUCC
AUUCAGGUGAAAAGCAGAUUCAGAUUGUUAAUUUUAAGCAGAUUUAUUAUACAGUCAGCGUAGACGCUGUUAAA-
AAUCCAGGAG
AUGUGUUUCAAGAUACUGUAACGGUAGAGGAUUUAAAACAGAGAGGAAUUUCUGCAGAGCGUCCUUUGGUCUAU-
AUUUCGAGUG
UUGCUUAUGGGCGCCAAGUCUAUCUCAAGUUGGAAACCACGAGUAAGAGUGAUGAAGUAGAGGCUGCUUUUGAA-
GCUUUGAUAA
AAGGAGUCAAGGUAGCUCCUCAGACAGAGUGGAAGCAGAUUUUGGACAAUACAGAAGUGAAGGCGGUUAUUUUA-
GGGGGCGACC
CAAGUUCGGGUGCCCGAGUUGUAACAGGCAAGGUGGAUAUGGUAGAGGACUUGAUUCAAGAAGGCAGUCGCUUU-
ACAGCAGAUC
AUCCAGGCUUGCCGAUUUCCUAUACAACUUCUUUUUUACGUGACAAUGUAGUUGCGACCUUUCAAAACAGUACA-
GACUAUGUUG
AGACUAAGGUUACAGCUUACAGAAACGGAGAUUUACUGCUGGAUCAUAGUGGUGCCUAUGUUGCCCAAUAUUAU-
AUUACUUGGG
AUGAAUUAUCCUAUGAUCAUCAAGGUAAGGAAGUCUUGACUCCUAAGGCUUGGGACAGAAAUGGGCAGGAUUUG-
ACGGCUCACU
UUACCACUAGUAUUCCUUUAAAAGGGAAUGUUCGUAAUCUCUCUGUCAAAAUUAGAGAGUGUACCGGGCUUGCC-
UGGGAAUGGU
GGCGUACGGUUUAUGAAAAAACCGAUUUGCCACUAGUGCGUAAGCGGACGAUUUCUAUUUGGGGAACAACUCUC-
UAUCCUCAGG UAGAGGAUAAGGUAGAAAAUGACUAGGAGAGGAGAAUGCUUGCGACAAAAAGA
(SEQ ID NO: 5) Sp_Ply_D205R_NGM_nIgK (5'UTR, ORF, 3' UTR)
UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGA-
AGAAAUAUAA
GAGCCACCAUGGAAACCCCUGCCCAGCUGCUGUUCCUGCUGCUGCUGUGGCUGCCUGACACCACCGGCAUGGCC-
AACAAGGCCG
UGAACGACUUCAUCCUGGCCAUGAACUACGACAAGAAGAAGCUGCUGACCCACCAGGGCGAGAGCAUCGAGAAC-
AGAUUCAUCA
AAGAGGGCAACCAGCUGCCCGACGAGUUCGUCGUGAUCGAGCGGAAGAAGCGGAGCCUGAGCACCGACACCAGC-
GACAUCAGCG
UGACCGCCACCAACGACGCCAGACUGUAUCCUGGCGCUCUGCUGGUGGUGGACGAGACACUGCUGGAAAACAAC-
CCCAUCCUGC
UGGCCGUGGACAGAGCCCCCAUGACCUACAGCAUCGACCUGCCUGGCCUGGCCAGCAGCGAUAGCUUUCUGCAG-
GUGGAAGAUC
CCAGCAACAGCGCCGUGCGGGGAGCCGUGAAUGACCUGCUGGCUAAGUGGCACCAGGACUACGGCCAAGUGAAC-
AACGUGCCCG
CCAGAAUGCAGUACGAGAAGAUCACCGCCCACUCCAUGGAACAGCUGAAAGUGAAGUUCGGCAGCGACUUCGAG-
AAAACCGGCA
ACAGCCUGGACAUCGACUUCAACAGCGUGCACAGCGGCGAGAAGCAGAUCCAGAUCGUGAACUUCAAGCAGAUC-
UACUACACCG
UGUCCGUGCGGGCCGUGAAGAACCCUGGGGACGUGUUCCAGGAUACCGUGACCGUGGAAGAUCUGAAGCAGCGG-
GGCAUCAGCG
CCGAGAGGCCACUGGUGUACAUCAGCAGCGUGGCCUACGGCAGACAGGUGUACCUGAAGCUGGAAACCACCUCC-
AAGAGCGACG
AGGUGGAAGCCGCCUUCGAGGCCCUGAUCAAGGGCGUGAAAGUGGCCCCUCAGACCGAGUGGAAGCAGAUUCUG-
GACAACACCG
AAGUGAAAGCCGUGAUCCUGGGCGGCGACCCUUCUAGCGGAGCCAGAGUCGUGACAGGCAAGGUGGACAUGGUG-
GAAGAUCUGA
UCCAGGAAGGCAGCCGGUUCACCGCCGAUCACCCUGGCCUGCCUAUCAGCUACACCACAAGCUUUCUGAGAGAC-
AACGUGGUGG
CCACAUUCCAGAACUCCGCCGACUACGUGGAAACAAAAGUGACCGCCUACCGGAACGGCGAUCUGCUGCUGGAU-
CACUCCGGCG
CCUAUGUGGCCCAGUACUACAUCACCUGGGACGAGCUGAGCUACGAUCACCAGGGCAAAGAGGUGCUGACCCCC-
AAGGCCUGGG
ACAGAAACGGCCAGGAUCUGACAGCCCACUUCACAACCAGCAUCCCCCUGAAGGGCAACGUGCGGAACCUGAGC-
GUGAAGAUCA
GAGAGUGCACCGGACUGGCCUGGGAGUGGUGGCGGACCGUGUACGAAAAGACCGACCUGCCCCUCGUGCGGAAG-
CGGACCAUCU
CUAUCUGGGGCACCACGCUGUAUCCUCAGGUGGAAGAUAAGGUGGAAAACGACUGAUAAUAGGCUGGAGCCUCG-
GUGGCCAUGC
UUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAG-
UCUGAGUGGG CGGC(SEQ ID NO: 6) SP_Ply_L460D_NGM_nIgK (5' UTR, ORF,
3' UTR)
UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGA-
AGAAAUAUAA
GAGCCACCAUGGAAACCCCUGCCCAGCUGCUGUUCCUGCUGCUGCUGUGGCUGCCUGACACCACCGGCAUGGCC-
AACAAGGCCG
UGAACGACUUCAUCCUGGCCAUGAACUACGACAAGAAGAAGCUGCUGACCCACCAGGGCGAGAGCAUCGAGAAC-
AGAUUCAUCA
AAGAGGGCAACCAGCUGCCCGACGAGUUCGUCGUGAUCGAGCGGAAGAAGCGGAGCCUGAGCACCGACACCAGC-
GACAUCAGCG
UGACCGCCACCAACGACGCCAGACUGUAUCCUGGCGCUCUGCUGGUGGUGGACGAGACACUGCUGGAAAACAAC-
CCCAUCCUGC
UGGCCGUGGACAGAGCCCCCAUGACCUACAGCAUCGACCUGCCUGGCCUGGCCAGCAGCGAUAGCUUUCUGCAG-
GUGGAAGAUC
CCAGCAACAGCGCCGUGCGGGGAGCCGUGAAUGACCUGCUGGCUAAGUGGCACCAGGACUACGGCCAAGUGAAC-
AACGUGCCCG
CCAGAAUGCAGUACGAGAAGAUCACCGCCCACUCCAUGGAACAGCUGAAAGUGAAGUUCGGCAGCGACUUCGAG-
AAAACCGGCA
ACAGCCUGGACAUCGACUUCAACAGCGUGCACAGCGGCGAGAAGCAGAUCCAGAUCGUGAACUUCAAGCAGAUC-
UACUACACCG
UGUCCGUGGACGCCGUGAAGAACCCCGGGGACGUGUUCCAGGAUACCGUGACCGUGGAAGAUCUGAAGCAGCGG-
GGCAUCAGCG
CCGAGAGGCCACUGGUGUACAUCAGCAGCGUGGCCUACGGCAGACAGGUGUACCUGAAGCUGGAAACCACCUCC-
AAGAGCGACG
AGGUGGAAGCCGCCUUCGAGGCCCUGAUCAAGGGCGUGAAAGUGGCCCCUCAGACCGAGUGGAAGCAGAUUCUG-
GACAACACCG
AAGUGAAAGCCGUGAUCCUGGGCGGCGACCCUUCUAGCGGAGCCAGAGUCGUGACAGGCAAGGUGGACAUGGUG-
GAAGAUCUGA
UCCAGGAAGGCAGCCGGUUCACCGCCGAUCACCCUGGCCUGCCUAUCAGCUACACCACAAGCUUUCUGAGAGAC-
AACGUGGUGG
CCACAUUCCAGAACUCCGCCGACUACGUGGAAACAAAAGUGACCGCCUACCGGAACGGCGAUCUGCUGCUGGAU-
CACUCCGGCG
CCUAUGUGGCCCAGUACUACAUCACCUGGGACGAGCUGAGCUACGAUCACCAGGGCAAAGAGGUGCUGACCCCC-
AAGGCCUGGG
ACAGAAACGGCCAGGAUCUGACAGCCCACUUCACAACCAGCAUCCCCCUGAAGGGCAACGUGCGGAACCUGAGC-
GUGAAGAUCA
GAGAGUGCACCGGACUGGCCUGGGAGUGGUGGCGGACCGUGUACGAAAAGACCGACCUGCCCCUCGUGCGGAAG-
CGGACCAUCU
CUAUCUGGGGCACCACCGACUACCCCCAGGUGGAAGAUAAGGUGGAAAACGACUGAUAAUAGGCUGGAGCCUCG-
GUGGCCAUGC
UUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAG-
UCUGAGUGG GCGGC(SEQ ID NO: 7) SP_Ply_T65C_G293C_C428A_PlyD1_
NGM_nIgK (5' UTR, ORF, 3' UTR)
UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGA-
AGAAAUAUAA
GAGCCACCAUGGAAACCCCUGCCCAGCUGCUGUUCCUGCUGCUGCUGUGGCUGCCUGACACCACCGGCAUGGCC-
AACAAGGCCG
UGAACGACUUCAUCCUGGCCAUGAACUACGACAAGAAGAAGCUGCUGACCCACCAGGGCGAGAGCAUCGAGAAC-
AGAUUCAUCA
AAGAGGGCAACCAGCUGCCCGACGAGUUCGUCGUGAUCGAGCGGAAGAAGCGGAGCCUGAGCACCGACACCAGC-
GACAUCAGCG
UGACCGCCUGCAACGACGCCAGACUGUAUCCUGGCGCUCUGCUGGUGGUGGACGAGACACUGCUGGAAAACAAC-
CCCAUCCUGC
UGGCCGUGGACAGAGCCCCCAUGACCUACAGCAUCGACCUGCCUGGCCUGGCCAGCAGCGAUAGCUUUCUGCAG-
GUGGAAGAUC
CCAGCAACAGCGCCGUGCGGGGAGCCGUGAAUGACCUGCUGGCUAAGUGGCACCAGGACUACGGCCAAGUGAAC-
AACGUGCCCG
CCAGAAUGCAGUACGAGAAGAUCACCGCCCACUCCAUGGAACAGCUGAAAGUGAAGUUCGGCAGCGACUUCGAG-
AAAACCGGCA
ACAGCCUGGACAUCGACUUCAACAGCGUGCACAGCGGCGAGAAGCAGAUCCAGAUCGUGAACUUCAAGCAGAUC-
UACUACACCG
UGUCCGUGGACGCCGUGAAGAACCCCGGGGACGUGUUCCAGGAUACCGUGACCGUGGAAGAUCUGAAGCAGCGG-
GGCAUCAGCG
CCGAGAGGCCACUGGUGUACAUCAGCAGCGUGGCCUACGGCAGACAGGUGUACCUGAAGCUGGAAACCACCUCC-
AAGAGCGACG
AGGUGGAAGCCGCCUUCGAGGCCCUGAUCAAGGGCGUGAAAGUGGCCCCUCAGACCGAGUGGAAGCAGAUUCUG-
GACAACACCG
AAGUGAAAGCCGUGAUCCUGUGCGGCGACCCUUCUAGCGGAGCCAGAGUCGUGACAGGCAAGGUGGACAUGGUG-
GAAGAUCUGA
UCCAGGAAGGCAGCCGGUUCACCGCCGAUCACCCUGGCCUGCCUAUCAGCUACACCACAAGCUUUCUGAGAGAC-
AACGUGGUGG
CCACAUUCCAGAACUCCGCCGACUACGUGGAAACAAAAGUGACAGCCUACCGGAACGGCGAUCUGCUGCUGGAU-
CACUCCGGCG
CCUAUGUGGCCCAGUACUACAUCACCUGGGACGAGCUGAGCUACGAUCACCAGGGCAAAGAGGUGCUGACCCCC-
AAGGCCUGGG
ACAGAAACGGCCAGGAUCUGACAGCCCACUUCACAACCAGCAUCCCCCUGAAGGGCAACGUGCGGAACCUGAGC-
GUGAAGAUCA
GAGAAGCCACCGGACUGGCCUGGGAGUGGUGGCGGACAGUGUACGAAAAGACCGACCUGCCCCUCGUGCGGAAG-
CGGACCAUCU
CUAUCUGGGGCACCACGCUGUAUCCUCAGGUGGAAGAUAAGGUGGAAAACGACUGAUAAUAGGCUGGAGCCUCG-
GUGGCCAUGC
UUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAG-
UCUGAGUGGG CGGC(SEQ ID NO: 8) SP_Ply_D205R_nIgK (5' UTR, ORF, 3'
UTR)
UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGA-
AGAAAUAUAA
GAGCCACCAUGGAAACCCCUGCCCAGCUGCUGUUCCUGCUGCUGCUGUGGCUGCCUGACACCACCGGCAUGGCC-
AACAAGGCCG
UGAACGACUUCAUCCUGGCCAUGAACUACGACAAGAAGAAGCUGCUGACCCACCAGGGCGAGAGCAUCGAGAAC-
AGAUUCAUCA
AAGAGGGCAACCAGCUGCCCGACGAGUUCGUCGUGAUCGAGCGGAAGAAGCGGAGCCUGAGCACCAACACCAGC-
GACAUCAGCG
UGACCGCCACCAACGACAGCAGACUGUAUCCUGGCGCCCUGCUGGUGGUGGACGAGACACUGCUGGAAAACAAC-
CCCACCCUGC
UGGCCGUGGACAGAGCCCCUAUGACCUACAGCAUCGACCUGCCUGGCCUGGCCAGCAGCGAUAGCUUUCUGCAG-
GUGGAAGAUC
CCAGCAACAGCAGCGUGCGGGGAGCCGUGAAUGACCUGCUGGCUAAGUGGCACCAGGACUACGGCCAAGUGAAC-
AACGUGCCCG
CCAGAAUGCAGUACGAGAAGAUCACCGCCCACUCCAUGGAACAGCUGAAAGUGAAGUUCGGCAGCGACUUCGAG-
AAAACCGGCA
ACAGCCUGGACAUCGACUUCAACAGCGUGCACAGCGGCGAGAAGCAGAUCCAGAUCGUGAACUUCAAGCAGAUC-
UACUACACCG
UGUCCGUGCGGGCCGUGAAGAACCCUGGGGACGUGUUCCAGGAUACCGUGACCGUGGAAGAUCUGAAGCAGCGG-
GGCAUCAGCG
CCGAGAGGCCACUGGUGUACAUCAGCUCUGUGGCCUACGGCAGACAGGUGUACCUGAAGCUGGAAACCACCUCC-
AAGAGCGACG
AGGUGGAAGCCGCCUUCGAGGCCCUGAUCAAGGGCGUGAAAGUGGCCCCUCAGACCGAGUGGAAGCAGAUUCUG-
GACAACACCG
AAGUGAAAGCCGUGAUCCUGGGCGGCGACCCUUCUAGCGGAGCCAGAGUCGUGACAGGCAAGGUGGACAUGGUG-
GAAGAUCUGA
UCCAGGAAGGCAGCCGGUUCACCGCCGAUCACCCUGGCCUGCCUAUCAGCUACACCACAAGCUUUCUGAGAGAC-
AACGUGGUGG
CCACAUUCCAGAACAGCACCGACUACGUGGAAACAAAAGUGACCGCCUACCGGAACGGCGAUCUGCUGCUGGAU-
CACUCCGGCG
CCUACGUGGCCCAGUACUACAUCACCUGGGACGAGCUGAGCUACGAUCACCAGGGCAAAGAGGUGCUGACCCCC-
AAGGCCUGGG
ACAGAAACGGCCAGGAUCUGACAGCCCACUUCACAACCAGCAUCCCCCUGAAGGGCAACGUGCGGAACCUGAGC-
GUGAAGAUCA
GAGAGUGCACCGGACUGGCCUGGGAGUGGUGGCGGACCGUGUACGAAAAGACCGACCUGCCCCUCGUGCGGAAG-
CGGACCAUCU
CUAUCUGGGGCACCACGCUGUAUCCUCAGGUGGAAGAUAAGGUGGAAAACGACUGAUAAUAGGCUGGAGCCUCG-
GUGGCCAUGC
UUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAG-
UCUGAGUGGG CGGC(SEQ ID NO: 33) SP_Ply_L460D_nIgK (5' UTR, ORF, 3'
UTR)
UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGA-
AGAAAUAUAA
GAGCCACCAUGGAAACCCCUGCCCAGCUGCUGUUCCUGCUGCUGCUGUGGCUGCCUGACACCACCGGCAUGGCC-
AACAAGGCCG
UGAACGACUUCAUCCUGGCCAUGAACUACGACAAGAAGAAGCUGCUGACCCACCAGGGCGAGAGCAUCGAGAAC-
AGAUUCAUCA
AAGAGGGCAACCAGCUGCCCGACGAGUUCGUCGUGAUCGAGCGGAAGAAGCGGAGCCUGAGCACCAACACCAGC-
GACAUCAGCG
UGACCGCCACCAACGACAGCAGACUGUAUCCUGGCGCCCUGCUGGUGGUGGACGAGACACUGCUGGAAAACAAC-
CCCACCCUGC
UGGCCGUGGACAGAGCCCCUAUGACCUACAGCAUCGACCUGCCUGGCCUGGCCAGCAGCGAUAGCUUUCUGCAG-
GUGGAAGAUC
CCAGCAACAGCAGCGUGCGGGGAGCCGUGAAUGACCUGCUGGCUAAGUGGCACCAGGACUACGGCCAAGUGAAC-
AACGUGCCCG
CCAGAAUGCAGUACGAGAAGAUCACCGCCCACUCCAUGGAACAGCUGAAAGUGAAGUUCGGCAGCGACUUCGAG-
AAAACCGGCA
ACAGCCUGGACAUCGACUUCAACAGCGUGCACAGCGGCGAGAAGCAGAUCCAGAUCGUGAACUUCAAGCAGAUC-
UACUACACCG
UGUCCGUGGACGCCGUGAAGAACCCCGGGGACGUGUUCCAGGAUACCGUGACCGUGGAAGAUCUGAAGCAGCGG-
GGCAUCAGCG
CCGAGAGGCCACUGGUGUACAUCAGCUCUGUGGCCUACGGCAGACAGGUGUACCUGAAGCUGGAAACCACCUCC-
AAGAGCGACG
AGGUGGAAGCCGCCUUCGAGGCCCUGAUCAAGGGCGUGAAAGUGGCCCCUCAGACCGAGUGGAAGCAGAUUCUG-
GACAACACCG
AAGUGAAAGCCGUGAUCCUGGGCGGCGACCCUUCUAGCGGAGCCAGAGUCGUGACAGGCAAGGUGGACAUGGUG-
GAAGAUCUGA
UCCAGGAAGGCAGCCGGUUCACCGCCGAUCACCCUGGCCUGCCUAUCAGCUACACCACAAGCUUUCUGAGAGAC-
AACGUGGUGG
CCACAUUCCAGAACAGCACCGACUACGUGGAAACAAAAGUGACCGCCUACCGGAACGGCGAUCUGCUGCUGGAU-
CACUCCGGCG
CCUACGUGGCCCAGUACUACAUCACCUGGGACGAGCUGAGCUACGAUCACCAGGGCAAAGAGGUGCUGACCCCC-
AAGGCCUGGG
ACAGAAACGGCCAGGAUCUGACAGCCCACUUCACAACCAGCAUCCCCCUGAAGGGCAACGUGCGGAACCUGAGC-
GUGAAGAUCA
GAGAGUGCACCGGACUGGCCUGGGAGUGGUGGCGGACCGUGUACGAAAAGACCGACCUGCCCCUCGUGCGGAAG-
CGGACCAUCU
CUAUCUGGGGCACCACCGAUUACCCCCAGGUGGAAGAUAAGGUGGAAAACGACUGAUAAUAGGCUGGAGCCUCG-
GUGGCCAUGC
UUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAG-
UCUGAGUGGG CGGC(SEQ ID NO: 34) SP_Ply_T65C_G293C_C428A_PlyD1_nIgK
(5' UTR, ORF, 3' UTR)
UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGA-
AGAAAUAUAA
GAGCCACCAUGGAAACCCCUGCCCAGCUGCUGUUCCUGCUGCUGCUGUGGCUGCCUGACACCACCGGCAUGGCC-
AACAAGGCCG
UGAACGACUUCAUCCUGGCCAUGAACUACGACAAGAAGAAGCUGCUGACCCACCAGGGCGAGAGCAUCGAGAAC-
AGAUUCAUCA
AAGAGGGCAACCAGCUGCCCGACGAGUUCGUCGUGAUCGAGCGGAAGAAGCGGAGCCUGAGCACCAACACCAGC-
GACAUCAGCG
UGACCGCCUGCAACGACAGCAGACUGUAUCCUGGCGCCCUGCUGGUGGUGGACGAGACACUGCUGGAAAACAAC-
CCCACCCUGC
UGGCCGUGGACAGAGCCCCUAUGACCUACAGCAUCGACCUGCCUGGCCUGGCCAGCAGCGAUAGCUUUCUGCAG-
GUGGAAGAUC
CCAGCAACAGCAGCGUGCGGGGAGCCGUGAAUGACCUGCUGGCUAAGUGGCACCAGGACUACGGCCAAGUGAAC-
AACGUGCCCG
CCAGAAUGCAGUACGAGAAGAUCACCGCCCACUCCAUGGAACAGCUGAAAGUGAAGUUCGGCAGCGACUUCGAG-
AAAACCGGCA
ACAGCCUGGACAUCGACUUCAACAGCGUGCACAGCGGCGAGAAGCAGAUCCAGAUCGUGAACUUCAAGCAGAUC-
UACUACACCG
UGUCCGUGGACGCCGUGAAGAACCCCGGGGACGUGUUCCAGGAUACCGUGACCGUGGAAGAUCUGAAGCAGCGG-
GGCAUCAGCG
CCGAGAGGCCACUGGUGUACAUCAGCUCUGUGGCCUACGGCAGACAGGUGUACCUGAAGCUGGAAACCACCUCC-
AAGAGCGACG
AGGUGGAAGCCGCCUUCGAGGCCCUGAUCAAGGGCGUGAAAGUGGCCCCUCAGACCGAGUGGAAGCAGAUUCUG-
GACAACACCG
AAGUGAAAGCCGUGAUCCUGUGCGGCGACCCUUCUAGCGGAGCCAGAGUCGUGACAGGCAAGGUGGACAUGGUG-
GAAGAUCUGA
UCCAGGAAGGCAGCCGGUUCACCGCCGAUCACCCUGGCCUGCCUAUCAGCUACACCACAAGCUUUCUGAGAGAC-
AACGUGGUGG
CCACAUUCCAGAACAGCACCGACUACGUGGAAACAAAAGUGACAGCCUACCGGAACGGCGAUCUGCUGCUGGAU-
CACUCCGGCG
CCUACGUGGCCCAGUACUACAUCACCUGGGACGAGCUGAGCUACGAUCACCAGGGCAAAGAGGUGCUGACCCCC-
AAGGCCUGGG
ACAGAAACGGCCAGGAUCUGACAGCCCACUUCACAACCAGCAUCCCCCUGAAGGGCAACGUGCGGAACCUGAGC-
GUGAAGAUCA
GAGAAGCCACCGGACUGGCCUGGGAGUGGUGGCGGACAGUGUACGAAAAGACCGACCUGCCCCUCGUGCGGAAG-
CGGACCAUCU
CUAUCUGGGGCACCACGCUGUAUCCUCAGGUGGAAGAUAAGGUGGAAAACGACUGAUAAUAGGCUGGAGCCUCG-
GUGGCCAUGC
UUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAG-
UCUGAGUGGG CGGC(SEQ ID NO: 35) Sp_Ply_D205R_NGM_nIgK (ORF)
AUGGAAACCCCUGCCCAGCUGCUGUUCCUGCUGCUGCUGUGGCUGCCUGACACCACCGGCAUGGCCAACAAGGC-
CGUGAACGAC
UUCAUCCUGGCCAUGAACUACGACAAGAAGAAGCUGCUGACCCACCAGGGCGAGAGCAUCGAGAACAGAUUCAU-
CAAAGAGGGC
AACCAGCUGCCCGACGAGUUCGUCGUGAUCGAGCGGAAGAAGCGGAGCCUGAGCACCGACACCAGCGACAUCAG-
CGUGACCGCC
ACCAACGACGCCAGACUGUAUCCUGGCGCUCUGCUGGUGGUGGACGAGACACUGCUGGAAAACAACCCCAUCCU-
GCUGGCCGUG
GACAGAGCCCCCAUGACCUACAGCAUCGACCUGCCUGGCCUGGCCAGCAGCGAUAGCUUUCUGCAGGUGGAAGA-
UCCCAGCAAC
AGCGCCGUGCGGGGAGCCGUGAAUGACCUGCUGGCUAAGUGGCACCAGGACUACGGCCAAGUGAACAACGUGCC-
CGCCAGAAUG
CAGUACGAGAAGAUCACCGCCCACUCCAUGGAACAGCUGAAAGUGAAGUUCGGCAGCGACUUCGAGAAAACCGG-
CAACAGCCUG
GACAUCGACUUCAACAGCGUGCACAGCGGCGAGAAGCAGAUCCAGAUCGUGAACUUCAAGCAGAUCUACUACAC-
CGUGUCCGUG
CGGGCCGUGAAGAACCCUGGGGACGUGUUCCAGGAUACCGUGACCGUGGAAGAUCUGAAGCAGCGGGGCAUCAG-
CGCCGAGAGG
CCACUGGUGUACAUCAGCAGCGUGGCCUACGGCAGACAGGUGUACCUGAAGCUGGAAACCACCUCCAAGAGCGA-
CGAGGUGGAA
GCCGCCUUCGAGGCCCUGAUCAAGGGCGUGAAAGUGGCCCCUCAGACCGAGUGGAAGCAGAUUCUGGACAACAC-
CGAAGUGAAA
GCCGUGAUCCUGGGCGGCGACCCUUCUAGCGGAGCCAGAGUCGUGACAGGCAAGGUGGACAUGGUGGAAGAUCU-
GAUCCAGGAA
GGCAGCCGGUUCACCGCCGAUCACCCUGGCCUGCCUAUCAGCUACACCACAAGCUUUCUGAGAGACAACGUGGU-
GGCCACAUUC
CAGAACUCCGCCGACUACGUGGAAACAAAAGUGACCGCCUACCGGAACGGCGAUCUGCUGCUGGAUCACUCCGG-
CGCCUAUGUG
GCCCAGUACUACAUCACCUGGGACGAGCUGAGCUACGAUCACCAGGGCAAAGAGGUGCUGACCCCCAAGGCCUG-
GGACAGAAAC
GGCCAGGAUCUGACAGCCCACUUCACAACCAGCAUCCCCCUGAAGGGCAACGUGCGGAACCUGAGCGUGAAGAU-
CAGAGAGUGC
ACCGGACUGGCCUGGGAGUGGUGGCGGACCGUGUACGAAAAGACCGACCUGCCCCUCGUGCGGAAGCGGACCAU-
CUCUAUCUGG GGCACCACGCUGUAUCCUCAGGUGGAAGAUAAGGUGGAAAACGAC(SEQ ID NO:
62) SP_Ply_L460D_NGM_nIgK (ORF)
AUGGAAACCCCUGCCCAGCUGCUGUUCCUGCUGCUGCUGUGGCUGCCUGACACCACCGGCAUGGCCAACAAGGC-
CGUGAACGAC
UUCAUCCUGGCCAUGAACUACGACAAGAAGAAGCUGCUGACCCACCAGGGCGAGAGCAUCGAGAACAGAUUCAU-
CAAAGAGGGC
AACCAGCUGCCCGACGAGUUCGUCGUGAUCGAGCGGAAGAAGCGGAGCCUGAGCACCGACACCAGCGACAUCAG-
CGUGACCGCC
ACCAACGACGCCAGACUGUAUCCUGGCGCUCUGCUGGUGGUGGACGAGACACUGCUGGAAAACAACCCCAUCCU-
GCUGGCCGUG
GACAGAGCCCCCAUGACCUACAGCAUCGACCUGCCUGGCCUGGCCAGCAGCGAUAGCUUUCUGCAGGUGGAAGA-
UCCCAGCAAC
AGCGCCGUGCGGGGAGCCGUGAAUGACCUGCUGGCUAAGUGGCACCAGGACUACGGCCAAGUGAACAACGUGCC-
CGCCAGAAUG
CAGUACGAGAAGAUCACCGCCCACUCCAUGGAACAGCUGAAAGUGAAGUUCGGCAGCGACUUCGAGAAAACCGG-
CAACAGCCUG
GACAUCGACUUCAACAGCGUGCACAGCGGCGAGAAGCAGAUCCAGAUCGUGAACUUCAAGCAGAUCUACUACAC-
CGUGUCCGUG
GACGCCGUGAAGAACCCCGGGGACGUGUUCCAGGAUACCGUGACCGUGGAAGAUCUGAAGCAGCGGGGCAUCAG-
CGCCGAGAGG
CCACUGGUGUACAUCAGCAGCGUGGCCUACGGCAGACAGGUGUACCUGAAGCUGGAAACCACCUCCAAGAGCGA-
CGAGGUGGAA
GCCGCCUUCGAGGCCCUGAUCAAGGGCGUGAAAGUGGCCCCUCAGACCGAGUGGAAGCAGAUUCUGGACAACAC-
CGAAGUGAAA
GCCGUGAUCCUGGGCGGCGACCCUUCUAGCGGAGCCAGAGUCGUGACAGGCAAGGUGGACAUGGUGGAAGAUCU-
GAUCCAGGAA
GGCAGCCGGUUCACCGCCGAUCACCCUGGCCUGCCUAUCAGCUACACCACAAGCUUUCUGAGAGACAACGUGGU-
GGCCACAUUC
CAGAACUCCGCCGACUACGUGGAAACAAAAGUGACCGCCUACCGGAACGGCGAUCUGCUGCUGGAUCACUCCGG-
CGCCUAUGUG
GCCCAGUACUACAUCACCUGGGACGAGCUGAGCUACGAUCACCAGGGCAAAGAGGUGCUGACCCCCAAGGCCUG-
GGACAGAAAC
GGCCAGGAUCUGACAGCCCACUUCACAACCAGCAUCCCCCUGAAGGGCAACGUGCGGAACCUGAGCGUGAAGAU-
CAGAGAGUGC
ACCGGACUGGCCUGGGAGUGGUGGCGGACCGUGUACGAAAAGACCGACCUGCCCCUCGUGCGGAAGCGGACCAU-
CUCUAUCUGG GGCACCACCGACUACCCCCAGGUGGAAGAUAAGGUGGAAAACGAC(SEQ ID NO:
63) SP_Ply_T65C_G293C_C428A_PlyD1_ NGM_nIgK (ORF)
AUGGAAACCCCUGCCCAGCUGCUGUUCCUGCUGCUGCUGUGGCUGCCUGACACCACCGGCAUGGCCAACAAGGC-
CGUGAACGAC
UUCAUCCUGGCCAUGAACUACGACAAGAAGAAGCUGCUGACCCACCAGGGCGAGAGCAUCGAGAACAGAUUCAU-
CAAAGAGGGC
AACCAGCUGCCCGACGAGUUCGUCGUGAUCGAGCGGAAGAAGCGGAGCCUGAGCACCGACACCAGCGACAUCAG-
CGUGACCGCC
UGCAACGACGCCAGACUGUAUCCUGGCGCUCUGCUGGUGGUGGACGAGACACUGCUGGAAAACAACCCCAUCCU-
GCUGGCCGUG
GACAGAGCCCCCAUGACCUACAGCAUCGACCUGCCUGGCCUGGCCAGCAGCGAUAGCUUUCUGCAGGUGGAAGA-
UCCCAGCAAC
AGCGCCGUGCGGGGAGCCGUGAAUGACCUGCUGGCUAAGUGGCACCAGGACUACGGCCAAGUGAACAACGUGCC-
CGCCAGAAUG
CAGUACGAGAAGAUCACCGCCCACUCCAUGGAACAGCUGAAAGUGAAGUUCGGCAGCGACUUCGAGAAAACCGG-
CAACAGCCUG
GACAUCGACUUCAACAGCGUGCACAGCGGCGAGAAGCAGAUCCAGAUCGUGAACUUCAAGCAGAUCUACUACAC-
CGUGUCCGUG
GACGCCGUGAAGAACCCCGGGGACGUGUUCCAGGAUACCGUGACCGUGGAAGAUCUGAAGCAGCGGGGCAUCAG-
CGCCGAGAGG
CCACUGGUGUACAUCAGCAGCGUGGCCUACGGCAGACAGGUGUACCUGAAGCUGGAAACCACCUCCAAGAGCGA-
CGAGGUGGAA
GCCGCCUUCGAGGCCCUGAUCAAGGGCGUGAAAGUGGCCCCUCAGACCGAGUGGAAGCAGAUUCUGGACAACAC-
CGAAGUGAAA
GCCGUGAUCCUGUGCGGCGACCCUUCUAGCGGAGCCAGAGUCGUGACAGGCAAGGUGGACAUGGUGGAAGAUCU-
GAUCCAGGAA
GGCAGCCGGUUCACCGCCGAUCACCCUGGCCUGCCUAUCAGCUACACCACAAGCUUUCUGAGAGACAACGUGGU-
GGCCACAUUC
CAGAACUCCGCCGACUACGUGGAAACAAAAGUGACAGCCUACCGGAACGGCGAUCUGCUGCUGGAUCACUCCGG-
CGCCUAUGUG
GCCCAGUACUACAUCACCUGGGACGAGCUGAGCUACGAUCACCAGGGCAAAGAGGUGCUGACCCCCAAGGCCUG-
GGACAGAAAC
GGCCAGGAUCUGACAGCCCACUUCACAACCAGCAUCCCCCUGAAGGGCAACGUGCGGAACCUGAGCGUGAAGAU-
CAGAGAAGCC
ACCGGACUGGCCUGGGAGUGGUGGCGGACAGUGUACGAAAAGACCGACCUGCCCCUCGUGCGGAAGCGGACCAU-
CUCUAUCUGG GGCACCACGCUGUAUCCUCAGGUGGAAGAUAAGGUGGAAAACGAC(SEQ ID NO:
64) SP_Ply_D205R_nIgK (ORF)
AUGGAAACCCCUGCCCAGCUGCUGUUCCUGCUGCUGCUGUGGCUGCCUGACACCACCGGCAUGGCCAACAAGGC-
CGUGAACGAC
UUCAUCCUGGCCAUGAACUACGACAAGAAGAAGCUGCUGACCCACCAGGGCGAGAGCAUCGAGAACAGAUUCAU-
CAAAGAGGGC
AACCAGCUGCCCGACGAGUUCGUCGUGAUCGAGCGGAAGAAGCGGAGCCUGAGCACCAACACCAGCGACAUCAG-
CGUGACCGCC
ACCAACGACAGCAGACUGUAUCCUGGCGCCCUGCUGGUGGUGGACGAGACACUGCUGGAAAACAACCCCACCCU-
GCUGGCCGUG
GACAGAGCCCCUAUGACCUACAGCAUCGACCUGCCUGGCCUGGCCAGCAGCGAUAGCUUUCUGCAGGUGGAAGA-
UCCCAGCAAC
AGCAGCGUGCGGGGAGCCGUGAAUGACCUGCUGGCUAAGUGGCACCAGGACUACGGCCAAGUGAACAACGUGCC-
CGCCAGAAUG
CAGUACGAGAAGAUCACCGCCCACUCCAUGGAACAGCUGAAAGUGAAGUUCGGCAGCGACUUCGAGAAAACCGG-
CAACAGCCUG
GACAUCGACUUCAACAGCGUGCACAGCGGCGAGAAGCAGAUCCAGAUCGUGAACUUCAAGCAGAUCUACUACAC-
CGUGUCCGUG
CGGGCCGUGAAGAACCCUGGGGACGUGUUCCAGGAUACCGUGACCGUGGAAGAUCUGAAGCAGCGGGGCAUCAG-
CGCCGAGAGG
CCACUGGUGUACAUCAGCUCUGUGGCCUACGGCAGACAGGUGUACCUGAAGCUGGAAACCACCUCCAAGAGCGA-
CGAGGUGGAA
GCCGCCUUCGAGGCCCUGAUCAAGGGCGUGAAAGUGGCCCCUCAGACCGAGUGGAAGCAGAUUCUGGACAACAC-
CGAAGUGAAA
GCCGUGAUCCUGGGCGGCGACCCUUCUAGCGGAGCCAGAGUCGUGACAGGCAAGGUGGACAUGGUGGAAGAUCU-
GAUCCAGGAA
GGCAGCCGGUUCACCGCCGAUCACCCUGGCCUGCCUAUCAGCUACACCACAAGCUUUCUGAGAGACAACGUGGU-
GGCCACAUUC
CAGAACAGCACCGACUACGUGGAAACAAAAGUGACCGCCUACCGGAACGGCGAUCUGCUGCUGGAUCACUCCGG-
CGCCUACGUG
GCCCAGUACUACAUCACCUGGGACGAGCUGAGCUACGAUCACCAGGGCAAAGAGGUGCUGACCCCCAAGGCCUG-
GGACAGAAAC
GGCCAGGAUCUGACAGCCCACUUCACAACCAGCAUCCCCCUGAAGGGCAACGUGCGGAACCUGAGCGUGAAGAU-
CAGAGAGUGC
ACCGGACUGGCCUGGGAGUGGUGGCGGACCGUGUACGAAAAGACCGACCUGCCCCUCGUGCGGAAGCGGACCAU-
CUCUAUCUGG GGCACCACGCUGUAUCCUCAGGUGGAAGAUAAGGUGGAAAACGAC(SEQ ID NO:
65) SP_Ply_L460D_nIgK (ORF)
AUGGAAACCCCUGCCCAGCUGCUGUUCCUGCUGCUGCUGUGGCUGCCUGACACCACCGGCAUGGCCAACAAGGC-
CGUGAACGAC
UUCAUCCUGGCCAUGAACUACGACAAGAAGAAGCUGCUGACCCACCAGGGCGAGAGCAUCGAGAACAGAUUCAU-
CAAAGAGGGC
AACCAGCUGCCCGACGAGUUCGUCGUGAUCGAGCGGAAGAAGCGGAGCCUGAGCACCAACACCAGCGACAUCAG-
CGUGACCGCC
ACCAACGACAGCAGACUGUAUCCUGGCGCCCUGCUGGUGGUGGACGAGACACUGCUGGAAAACAACCCCACCCU-
GCUGGCCGUG
GACAGAGCCCCUAUGACCUACAGCAUCGACCUGCCUGGCCUGGCCAGCAGCGAUAGCUUUCUGCAGGUGGAAGA-
UCCCAGCAAC
AGCAGCGUGCGGGGAGCCGUGAAUGACCUGCUGGCUAAGUGGCACCAGGACUACGGCCAAGUGAACAACGUGCC-
CGCCAGAAUG
CAGUACGAGAAGAUCACCGCCCACUCCAUGGAACAGCUGAAAGUGAAGUUCGGCAGCGACUUCGAGAAAACCGG-
CAACAGCCUG
GACAUCGACUUCAACAGCGUGCACAGCGGCGAGAAGCAGAUCCAGAUCGUGAACUUCAAGCAGAUCUACUACAC-
CGUGUCCGUG
GACGCCGUGAAGAACCCCGGGGACGUGUUCCAGGAUACCGUGACCGUGGAAGAUCUGAAGCAGCGGGGCAUCAG-
CGCCGAGAGG
CCACUGGUGUACAUCAGCUCUGUGGCCUACGGCAGACAGGUGUACCUGAAGCUGGAAACCACCUCCAAGAGCGA-
CGAGGUGGAA
GCCGCCUUCGAGGCCCUGAUCAAGGGCGUGAAAGUGGCCCCUCAGACCGAGUGGAAGCAGAUUCUGGACAACAC-
CGAAGUGAAA
GCCGUGAUCCUGGGCGGCGACCCUUCUAGCGGAGCCAGAGUCGUGACAGGCAAGGUGGACAUGGUGGAAGAUCU-
GAUCCAGGAA
GGCAGCCGGUUCACCGCCGAUCACCCUGGCCUGCCUAUCAGCUACACCACAAGCUUUCUGAGAGACAACGUGGU-
GGCCACAUUC
CAGAACAGCACCGACUACGUGGAAACAAAAGUGACCGCCUACCGGAACGGCGAUCUGCUGCUGGAUCACUCCGG-
CGCCUACGUG
GCCCAGUACUACAUCACCUGGGACGAGCUGAGCUACGAUCACCAGGGCAAAGAGGUGCUGACCCCCAAGGCCUG-
GGACAGAAAC
GGCCAGGAUCUGACAGCCCACUUCACAACCAGCAUCCCCCUGAAGGGCAACGUGCGGAACCUGAGCGUGAAGAU-
CAGAGAGUGC
ACCGGACUGGCCUGGGAGUGGUGGCGGACCGUGUACGAAAAGACCGACCUGCCCCUCGUGCGGAAGCGGACCAU-
CUCUAUCUGG GGCACCACCGAUUACCCCCAGGUGGAAGAUAAGGUGGAAAACGAC(SEQ ID NO:
66) SP_Ply_T65C_G293C_C428A_PlyD1_nIgK (ORF)
AUGGAAACCCCUGCCCAGCUGCUGUUCCUGCUGCUGCUGUGGCUGCCUGACACCACCGGCAUGGCCAACAAGGC-
CGUGAACGAC
UUCAUCCUGGCCAUGAACUACGACAAGAAGAAGCUGCUGACCCACCAGGGCGAGAGCAUCGAGAACAGAUUCAU-
CAAAGAGGGC
AACCAGCUGCCCGACGAGUUCGUCGUGAUCGAGCGGAAGAAGCGGAGCCUGAGCACCAACACCAGCGACAUCAG-
CGUGACCGCC
UGCAACGACAGCAGACUGUAUCCUGGCGCCCUGCUGGUGGUGGACGAGACACUGCUGGAAAACAACCCCACCCU-
GCUGGCCGUG
GACAGAGCCCCUAUGACCUACAGCAUCGACCUGCCUGGCCUGGCCAGCAGCGAUAGCUUUCUGCAGGUGGAAGA-
UCCCAGCAAC
AGCAGCGUGCGGGGAGCCGUGAAUGACCUGCUGGCUAAGUGGCACCAGGACUACGGCCAAGUGAACAACGUGCC-
CGCCAGAAUG
CAGUACGAGAAGAUCACCGCCCACUCCAUGGAACAGCUGAAAGUGAAGUUCGGCAGCGACUUCGAGAAAACCGG-
CAACAGCCUG
GACAUCGACUUCAACAGCGUGCACAGCGGCGAGAAGCAGAUCCAGAUCGUGAACUUCAAGCAGAUCUACUACAC-
CGUGUCCGUG
GACGCCGUGAAGAACCCCGGGGACGUGUUCCAGGAUACCGUGACCGUGGAAGAUCUGAAGCAGCGGGGCAUCAG-
CGCCGAGAGG
CCACUGGUGUACAUCAGCUCUGUGGCCUACGGCAGACAGGUGUACCUGAAGCUGGAAACCACCUCCAAGAGCGA-
CGAGGUGGAA
GCCGCCUUCGAGGCCCUGAUCAAGGGCGUGAAAGUGGCCCCUCAGACCGAGUGGAAGCAGAUUCUGGACAACAC-
CGAAGUGAAA
GCCGUGAUCCUGUGCGGCGACCCUUCUAGCGGAGCCAGAGUCGUGACAGGCAAGGUGGACAUGGUGGAAGAUCU-
GAUCCAGGAA
GGCAGCCGGUUCACCGCCGAUCACCCUGGCCUGCCUAUCAGCUACACCACAAGCUUUCUGAGAGACAACGUGGU-
GGCCACAUUC
CAGAACAGCACCGACUACGUGGAAACAAAAGUGACAGCCUACCGGAACGGCGAUCUGCUGCUGGAUCACUCCGG-
CGCCUACGUG
GCCCAGUACUACAUCACCUGGGACGAGCUGAGCUACGAUCACCAGGGCAAAGAGGUGCUGACCCCCAAGGCCUG-
GGACAGAAAC
GGCCAGGAUCUGACAGCCCACUUCACAACCAGCAUCCCCCUGAAGGGCAACGUGCGGAACCUGAGCGUGAAGAU-
CAGAGAAGCC
ACCGGACUGGCCUGGGAGUGGUGGCGGACAGUGUACGAAAAGACCGACCUGCCCCUCGUGCGGAAGCGGACCAU-
CUCUAUCUGG GGCACCACGCUGUAUCCUCAGGUGGAAGAUAAGGUGGAAAACGAC(SEQ ID NO:
67)
TABLE-US-00016 TABLE 2 Pneumolysin Amino Acid Sequences Descripti
Sequence SEQ ID NO: pneumolysin [Streptococcus pneumoniae];
Accession No. AJS15225.1
MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTNTSDISVTATNDSRLYPGA-
LLVVDETLL
ENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQYEKITAHS-
MEQLKVKFG
SDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQRGISAERPLVYISSV-
AYGRQVYLK
LETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQEGSRFTAD-
HPGLPISYT
TSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWNELSYDHQGKEVLTPKAWDRNGQDLTA-
HFTTSIPLK GNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEND
(SEQ ID NO: 9) 4ZGH: A|PDBID|CHAIN|SEQUENCE
HHHHHHGSANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTSTSDISVTATND-
SRLYPGALL
VVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQY-
EKITAHSME
QLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVRNPGDVFQDTVTVEDLKQRGISAERP-
LVYISSVAY
GRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVDDLIQE-
GSRFTADHP
GLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDR-
NGQDLTAHF
TTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEN (SEQ
ID NO: 10) VAR1_D102F
HHHHHHGSANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTSTSDISVTATND-
SRLYPGALL
VVDETLLENNPTLLAVDRAPMTYSIFLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQY-
EKITAHSME
QLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVRNPGDVFQDTVTVEDLKQRGISAERP-
LVYISSVAY
GRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVDDLIQE-
GSRFTADHP
GLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDR-
NGQDLTAHF
TTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEN (SEQ
ID NO: 11) VAR2_D102M
HHHHHHGSANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTSTSDISVTATND-
SRLYPGALL
VVDETLLENNPTLLAVDRAPMTYSIMLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQY-
EKITAHSME
QLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVRNPGDVFQDTVTVEDLKQRGISAERP-
LVYISSVAY
GRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVDDLIQE-
GSRFTADHP
GLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDR-
NGQDLTAHF
TTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEN (SEQ
ID NO: 12) VAR3_A107M
HHHHHHGSANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTSTSDISVTATND-
SRLYPGALL
VVDETLLENNPTLLAVDRAPMTYSIDLPGLMSSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQY-
EKITAHSME
QLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVRNPGDVFQDTVTVEDLKQRGISAERP-
LVYISSVAY
GRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVDDLIQE-
GSRFTADHP
GLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDR-
NGQDLTAHF
TTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEN (SEQ
ID NO: 13) VAR4_A107Q
HHHHHHGSANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTSTSDISVTATND-
SRLYPGALL
VVDETLLENNPTLLAVDRAPMTYSIDLPGLQSSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQY-
EKITAHSME
QLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVRNPGDVFQDTVTVEDLKQRGISAERP-
LVYISSVAY
GRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVDDLIQE-
GSRFTADHP
GLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDR-
NGQDLTAHF
TTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEN (SEQ
ID NO: 14) VARS_N120F
HHHHHHGSANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTSTSDISVTATND-
SRLYPGALL
VVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSFSSVRGAVNDLLAKWHQDYGQVNNVPARMQY-
EKITAHSME
QLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVRNPGDVFQDTVTVEDLKQRGISAERP-
LVYISSVAY
GRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVDDLIQE-
GSRFTADHP
GLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDR-
NGQDLTAHF
TTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEN (SEQ
ID NO: 15) VAR6_E187R
HHHHHHGSANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTSTSDISVTATND-
SRLYPGALL
VVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQY-
EKITAHSME
QLKVKFGSDFEKTGNSLDIDFNSVHSGRKQIQIVNFKQIYYTVSVDAVRNPGDVFQDTVTVEDLKQRGISAERP-
LVYISSVAY
GRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVDDLIQE-
GSRFTADHP
GLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDR-
NGQDLTAHF
TTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEN (SEQ
ID NO: 16) VAR7_E187L
HHHHHHGSANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTSTSDISVTATND-
SRLYPGALL
VVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQY-
EKITAHSME
QLKVKFGSDFEKTGNSLDIDFNSVHSGLKQIQIVNFKQIYYTVSVDAVRNPGDVFQDTVTVEDLKQRGISAERP-
LVYISSVAY
GRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVDDLIQE-
GSRFTADHP
GLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDR-
NGQDLTAHF
TTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEN (SEQ
ID NO: 17) VAR8_D102F_A107Q
HHHHHHGSANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTSTSDISVTATND-
SRLYPGALL
VVDETLLENNPTLLAVDRAPMTYSIFLPGLQSSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQY-
EKITAHSME
QLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVRNPGDVFQDTVTVEDLKQRGISAERP-
LVYISSVAY
GRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVDDLIQE-
GSRFTADHP
GLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDR-
NGQDLTAHF
TTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEN (SEQ
ID NO: 18) VAR9_D102F_N120F
HHHHHHGSANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTSTSDISVTATND-
SRLYPGALL
VVDETLLENNPTLLAVDRAPMTYSIFLPGLASSDSFLQVEDPSFSSVRGAVNDLLAKWHQDYGQVNNVPARMQY-
EKITAHSME
QLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVRNPGDVFQDTVTVEDLKQRGISAERP-
LVYISSVAY
GRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVDDLIQE-
GSRFTADHP
GLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDR-
NGQDLTAHF
TTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEN (SEQ
ID NO: 19) VAR10_A107Q_N120F
HHHHHHGSANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTSTSDISVTATND-
SRLYPGALL
VVDETLLENNPTLLAVDRAPMTYSIDLPGLQSSDSFLQVEDPSFSSVRGAVNDLLAKWHQDYGQVNNVPARMQY-
EKITAHSME
QLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVRNPGDVFQDTVTVEDLKQRGISAERP-
LVYISSVAY
GRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVDDLIQE-
GSRFTADHP
GLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDR-
NGQDLTAHF
TTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEN (SEQ
ID NO: 20) VAR11_D102F_A107Q_N120F
HHHHHHGSANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTSTSDISVTATND-
SRLYPGALL
VVDETLLENNPTLLAVDRAPMTYSIFLPGLQSSDSFLQVEDPSFSSVRGAVNDLLAKWHQDYGQVNNVPARMQY-
EKITAHSME
QLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVRNPGDVFQDTVTVEDLKQRGISAERP-
LVYISSVAY
GRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVDDLIQE-
GSRFTADHP
GLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDR-
NGQDLTAHF
TTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEN (SEQ
ID NO: 21) VAR12_D102F_A107Q_N120F_E187L
HHHHHHGSANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTSTSDISVTATND-
SRLYPGALL
VVDETLLENNPTLLAVDRAPMTYSIFLPGLQSSDSFLQVEDPSFSSVRGAVNDLLAKWHQDYGQVNNVPARMQY-
EKITAHSME
QLKVKFGSDFEKTGNSLDIDFNSVHSGLKQIQIVNFKQIYYTVSVDAVRNPGDVFQDTVTVEDLKQRGISAERP-
LVYISSVAY
GRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVDDLIQE-
GSRFTADHP
GLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDR-
NGQDLTAHF
TTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEN (SEQ
ID NO: 22) VAR13_D205F
HHHHHHGSANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTSTSDISVTATND-
SRLYPGALL
VVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQY-
EKITAHSME
QLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVFAVRNPGDVFQDTVTVEDLKQRGISAERP-
LVYISSVAY
GRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVDDLIQE-
GSRFTADHP
GLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDR-
NGQDLTAHF
TTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEN (SEQ
ID NO: 23) VAR14_D205P
HHHHHHGSANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTSTSDISVTATND-
SRLYPGALL
VVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQY-
EKITAHSME
QLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVPAVRNPGDVFQDTVTVEDLKQRGISAERP-
LVYISSVAY
GRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVDDLIQE-
GSRFTADHP
GLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDR-
NGQDLTAHF
TTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEN (SEQ
ID NO: 24) VAR15_G2931
HHHHHHGSANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTSTSDISVTATND-
SRLYPGALL
VVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQDYGQVNNVPARMQY-
EKITAHSME
QLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVRNPGDVFQDTVTVEDLKQRGISAERP-
LVYISSVAY
GRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILIGDPSSGARVVTGKVDMVDDLIQE-
GSRFTADHP
GLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDR-
NGQDLTAHF
TTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEN (SEQ
ID NO: 25) VAR16_N120F_D205P
HHHHHHGSANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLSTSTSDISVTATND-
SRLYPGALL
VVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSFSSVRGAVNDLLAKWHQDYGQVNNVPARMQY-
EKITAHSME
QLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVPAVRNPGDVFQDTVTVEDLKQRGISAERP-
LVYISSVAY
GRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVDDLIQE-
GSRFTADHP
GLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDR-
NGQDLTAHF
TTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVEN (SEQ
ID NO: 26) SP_Ply_D205R_NGM_nIgK
METPAQLLFLLLLWLPDTTGMANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLS-
TDTSDISVT
ATNDARLYPGALLVVDETLLENNPILLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSAVRGAVNDLLAKWHQD-
YGQVNNVPA
RMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVRAVKNPGDVFQDTVTV-
EDLKQRGIS
AERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVV-
TGKVDMVED
LIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSADYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDH-
QGKEVLTPK
AWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVE-
ND (SEQ ID NO: 27) SP_Ply_L460D_NGM_nIgK
METPAQLLFLLLLWLPDTTGMANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLS-
TDTSDISVT
ATNDARLYPGALLVVDETLLENNPILLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSAVRGAVNDLLAKWHQD-
YGQVNNVPA
RMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTV-
EDLKQRGIS
AERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVV-
TGKVDMVED
LIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSADYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDH-
QGKEVLTPK
AWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTDYPQVEDKVE-
ND (SEQ ID NO: 28) SP_Ply_T65C_G293C_C428A_PlyD1_NGM_nIgK
METPAQLLFLLLLWLPDTTGMANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLS-
TDTSDISVT
ACNDARLYPGALLVVDETLLENNPILLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSAVRGAVNDLLAKWHQD-
YGQVNNVPA
RMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTV-
EDLKQRGIS
AERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILCGDPSSGARVV-
TGKVDMVED
LIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSADYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDH-
QGKEVLTPK
AWDRNGQDLTAHFTTSIPLKGNVRNLSVKIREATGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVE-
ND (SEQ ID NO: 29) SP_Ply_D205R_nIgK
METPAQLLFLLLLWLPDTTGMANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLS-
TNTSDISVT
ATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQD-
YGQVNNVPA
RMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVRAVKNPGDVFQDTVTV-
EDLKQRGIS
AERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVV-
TGKVDMVED
LIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDH-
QGKEVLTPK
AWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVE-
ND (SEQ ID NO: 36) SP_Ply_L460D_nIgK
METPAQLLFLLLLWLPDTTGMANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLS-
TNTSDISVT
ATNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQD-
YGQVNNVPA
RMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTV-
EDLKQRGIS
AERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILGGDPSSGARVV-
TGKVDMVED
LIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDH-
QGKEVLTPK
AWDRNGQDLTAHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRKRTISIWGTTDYPQVEDKVE-
ND (SEQ ID NO: 37) SP_Ply_T65C_G293C_C428A_PlyD1_nIgK
METPAQLLFLLLLWLPDTTGMANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVVIERKKRSLS-
TNTSDISVT
ACNDSRLYPGALLVVDETLLENNPTLLAVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWHQD-
YGQVNNVPA
RMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDFNSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTV-
EDLKQRGIS
AERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGVKVAPQTEWKQILDNTEVKAVILCGDPSSGARVV-
TGKVDMVED
LIQEGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRNGDLLLDHSGAYVAQYYITWDELSYDH-
QGKEVLTPK
AWDRNGQDLTAHFTTSIPLKGNVRNLSVKIREATGLAWEWWRTVYEKTDLPLVRKRTISIWGTTLYPQVEDKVE-
ND (SEQ ID NO: 38)
EQUIVALENTS
[0537] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the disclosure described
herein. Such equivalents are intended to be encompassed by the
following claims.
[0538] All references, including patent documents, disclosed herein
are incorporated by reference in their entirety.
Sequence CWU 1
1
6911649DNAS.pneumoniae 1ttacaagacc aaccttgatt gacttagata aggtatttat
gttggataat acggttattc 60cgacttctta tctagccaga cggcgacgca atgtctcaga
agaattgtac gaggaaattt 120tggatcactt agtccaacca cggctgattt
cgctgaacaa gtctgagttt atgcaactca 180atccaggaac ttattaggag
gagaagatgg caaataaagc agtaaatgac tttatactag 240ctatgaatta
cgataaaaag aaactcttga cccatcaggg agaaagtatt gaaaatcgtt
300tcatcaaaga gggtaatcag ctacccgatg agtttgttgt tatcgaaaga
aagaagcgga 360gcttgtcgac aaatacaagt gatatttctg taacagctac
caacgacagt cgcctctatc 420ctggagcact tctcgtagtg gatgagacct
tgttagagaa taatcccact cttcttgcgg 480ttgatcgtgc tccgatgact
tatagtattg atttgcctgg tttggcaagt agcgatagct 540ttctccaagt
ggaagacccc agcaattcaa gtgttcgcgg agcggtaaac gatttgttgg
600ctaagtggca tcaagattat ggtcaggtca ataatgtccc agctagaatg
cagtatgaaa 660aaataacggc tcacagcatg gaacaactca aggtcaagtt
tggttctgac tttgaaaaga 720cagggaattc tcttgatatt gattttaact
ctgtccattc aggtgaaaag cagattcaga 780ttgttaattt taagcagatt
tattatacag tcagcgtaga cgctgttaaa aatccaggag 840atgtgtttca
agatactgta acggtagagg atttaaaaca gagaggaatt tctgcagagc
900gtcctttggt ctatatttcg agtgttgctt atgggcgcca agtctatctc
aagttggaaa 960ccacgagtaa gagtgatgaa gtagaggctg cttttgaagc
tttgataaaa ggagtcaagg 1020tagctcctca gacagagtgg aagcagattt
tggacaatac agaagtgaag gcggttattt 1080tagggggcga cccaagttcg
ggtgcccgag ttgtaacagg caaggtggat atggtagagg 1140acttgattca
agaaggcagt cgctttacag cagatcatcc aggcttgccg atttcctata
1200caacttcttt tttacgtgac aatgtagttg cgacctttca aaacagtaca
gactatgttg 1260agactaaggt tacagcttac agaaacggag atttactgct
ggatcatagt ggtgcctatg 1320ttgcccaata ttatattact tgggatgaat
tatcctatga tcatcaaggt aaggaagtct 1380tgactcctaa ggcttgggac
agaaatgggc aggatttgac ggctcacttt accactagta 1440ttcctttaaa
agggaatgtt cgtaatctct ctgtcaaaat tagagagtgt accgggcttg
1500cctgggaatg gtggcgtacg gtttatgaaa aaaccgattt gccactagtg
cgtaagcgga 1560cgatttctat ttggggaaca actctctatc ctcaggtaga
ggataaggta gaaaatgact 1620aggagaggag aatgcttgcg acaaaaaga
164921684DNAArtificial SequenceSynthetic polynucleotide 2tcaagctttt
ggaccctcgt acagaagcta atacgactca ctatagggaa ataagagaga 60aaagaagagt
aagaagaaat ataagagcca ccatggaaac ccctgcccag ctgctgttcc
120tgctgctgct gtggctgcct gacaccaccg gcatggccaa caaggccgtg
aacgacttca 180tcctggccat gaactacgac aagaagaagc tgctgaccca
ccagggcgag agcatcgaga 240acagattcat caaagagggc aaccagctgc
ccgacgagtt cgtcgtgatc gagcggaaga 300agcggagcct gagcaccgac
accagcgaca tcagcgtgac cgccaccaac gacgccagac 360tgtatcctgg
cgctctgctg gtggtggacg agacactgct ggaaaacaac cccatcctgc
420tggccgtgga cagagccccc atgacctaca gcatcgacct gcctggcctg
gccagcagcg 480atagctttct gcaggtggaa gatcccagca acagcgccgt
gcggggagcc gtgaatgacc 540tgctggctaa gtggcaccag gactacggcc
aagtgaacaa cgtgcccgcc agaatgcagt 600acgagaagat caccgcccac
tccatggaac agctgaaagt gaagttcggc agcgacttcg 660agaaaaccgg
caacagcctg gacatcgact tcaacagcgt gcacagcggc gagaagcaga
720tccagatcgt gaacttcaag cagatctact acaccgtgtc cgtgcgggcc
gtgaagaacc 780ctggggacgt gttccaggat accgtgaccg tggaagatct
gaagcagcgg ggcatcagcg 840ccgagaggcc actggtgtac atcagcagcg
tggcctacgg cagacaggtg tacctgaagc 900tggaaaccac ctccaagagc
gacgaggtgg aagccgcctt cgaggccctg atcaagggcg 960tgaaagtggc
ccctcagacc gagtggaagc agattctgga caacaccgaa gtgaaagccg
1020tgatcctggg cggcgaccct tctagcggag ccagagtcgt gacaggcaag
gtggacatgg 1080tggaagatct gatccaggaa ggcagccggt tcaccgccga
tcaccctggc ctgcctatca 1140gctacaccac aagctttctg agagacaacg
tggtggccac attccagaac tccgccgact 1200acgtggaaac aaaagtgacc
gcctaccgga acggcgatct gctgctggat cactccggcg 1260cctatgtggc
ccagtactac atcacctggg acgagctgag ctacgatcac cagggcaaag
1320aggtgctgac ccccaaggcc tgggacagaa acggccagga tctgacagcc
cacttcacaa 1380ccagcatccc cctgaagggc aacgtgcgga acctgagcgt
gaagatcaga gagtgcaccg 1440gactggcctg ggagtggtgg cggaccgtgt
acgaaaagac cgacctgccc ctcgtgcgga 1500agcggaccat ctctatctgg
ggcaccacgc tgtatcctca ggtggaagat aaggtggaaa 1560acgactgata
ataggctgga gcctcggtgg ccatgcttct tgccccttgg gcctcccccc
1620agcccctcct ccccttcctg cacccgtacc cccgtggtct ttgaataaag
tctgagtggg 1680cggc 168431684DNAArtificial SequenceSynthetic
polynucleotide 3tcaagctttt ggaccctcgt acagaagcta atacgactca
ctatagggaa ataagagaga 60aaagaagagt aagaagaaat ataagagcca ccatggaaac
ccctgcccag ctgctgttcc 120tgctgctgct gtggctgcct gacaccaccg
gcatggccaa caaggccgtg aacgacttca 180tcctggccat gaactacgac
aagaagaagc tgctgaccca ccagggcgag agcatcgaga 240acagattcat
caaagagggc aaccagctgc ccgacgagtt cgtcgtgatc gagcggaaga
300agcggagcct gagcaccgac accagcgaca tcagcgtgac cgccaccaac
gacgccagac 360tgtatcctgg cgctctgctg gtggtggacg agacactgct
ggaaaacaac cccatcctgc 420tggccgtgga cagagccccc atgacctaca
gcatcgacct gcctggcctg gccagcagcg 480atagctttct gcaggtggaa
gatcccagca acagcgccgt gcggggagcc gtgaatgacc 540tgctggctaa
gtggcaccag gactacggcc aagtgaacaa cgtgcccgcc agaatgcagt
600acgagaagat caccgcccac tccatggaac agctgaaagt gaagttcggc
agcgacttcg 660agaaaaccgg caacagcctg gacatcgact tcaacagcgt
gcacagcggc gagaagcaga 720tccagatcgt gaacttcaag cagatctact
acaccgtgtc cgtggacgcc gtgaagaacc 780ccggggacgt gttccaggat
accgtgaccg tggaagatct gaagcagcgg ggcatcagcg 840ccgagaggcc
actggtgtac atcagcagcg tggcctacgg cagacaggtg tacctgaagc
900tggaaaccac ctccaagagc gacgaggtgg aagccgcctt cgaggccctg
atcaagggcg 960tgaaagtggc ccctcagacc gagtggaagc agattctgga
caacaccgaa gtgaaagccg 1020tgatcctggg cggcgaccct tctagcggag
ccagagtcgt gacaggcaag gtggacatgg 1080tggaagatct gatccaggaa
ggcagccggt tcaccgccga tcaccctggc ctgcctatca 1140gctacaccac
aagctttctg agagacaacg tggtggccac attccagaac tccgccgact
1200acgtggaaac aaaagtgacc gcctaccgga acggcgatct gctgctggat
cactccggcg 1260cctatgtggc ccagtactac atcacctggg acgagctgag
ctacgatcac cagggcaaag 1320aggtgctgac ccccaaggcc tgggacagaa
acggccagga tctgacagcc cacttcacaa 1380ccagcatccc cctgaagggc
aacgtgcgga acctgagcgt gaagatcaga gagtgcaccg 1440gactggcctg
ggagtggtgg cggaccgtgt acgaaaagac cgacctgccc ctcgtgcgga
1500agcggaccat ctctatctgg ggcaccaccg actaccccca ggtggaagat
aaggtggaaa 1560acgactgata ataggctgga gcctcggtgg ccatgcttct
tgccccttgg gcctcccccc 1620agcccctcct ccccttcctg cacccgtacc
cccgtggtct ttgaataaag tctgagtggg 1680cggc 168441684DNAArtificial
SequenceSynthetic polynucleotide 4tcaagctttt ggaccctcgt acagaagcta
atacgactca ctatagggaa ataagagaga 60aaagaagagt aagaagaaat ataagagcca
ccatggaaac ccctgcccag ctgctgttcc 120tgctgctgct gtggctgcct
gacaccaccg gcatggccaa caaggccgtg aacgacttca 180tcctggccat
gaactacgac aagaagaagc tgctgaccca ccagggcgag agcatcgaga
240acagattcat caaagagggc aaccagctgc ccgacgagtt cgtcgtgatc
gagcggaaga 300agcggagcct gagcaccgac accagcgaca tcagcgtgac
cgcctgcaac gacgccagac 360tgtatcctgg cgctctgctg gtggtggacg
agacactgct ggaaaacaac cccatcctgc 420tggccgtgga cagagccccc
atgacctaca gcatcgacct gcctggcctg gccagcagcg 480atagctttct
gcaggtggaa gatcccagca acagcgccgt gcggggagcc gtgaatgacc
540tgctggctaa gtggcaccag gactacggcc aagtgaacaa cgtgcccgcc
agaatgcagt 600acgagaagat caccgcccac tccatggaac agctgaaagt
gaagttcggc agcgacttcg 660agaaaaccgg caacagcctg gacatcgact
tcaacagcgt gcacagcggc gagaagcaga 720tccagatcgt gaacttcaag
cagatctact acaccgtgtc cgtggacgcc gtgaagaacc 780ccggggacgt
gttccaggat accgtgaccg tggaagatct gaagcagcgg ggcatcagcg
840ccgagaggcc actggtgtac atcagcagcg tggcctacgg cagacaggtg
tacctgaagc 900tggaaaccac ctccaagagc gacgaggtgg aagccgcctt
cgaggccctg atcaagggcg 960tgaaagtggc ccctcagacc gagtggaagc
agattctgga caacaccgaa gtgaaagccg 1020tgatcctgtg cggcgaccct
tctagcggag ccagagtcgt gacaggcaag gtggacatgg 1080tggaagatct
gatccaggaa ggcagccggt tcaccgccga tcaccctggc ctgcctatca
1140gctacaccac aagctttctg agagacaacg tggtggccac attccagaac
tccgccgact 1200acgtggaaac aaaagtgaca gcctaccgga acggcgatct
gctgctggat cactccggcg 1260cctatgtggc ccagtactac atcacctggg
acgagctgag ctacgatcac cagggcaaag 1320aggtgctgac ccccaaggcc
tgggacagaa acggccagga tctgacagcc cacttcacaa 1380ccagcatccc
cctgaagggc aacgtgcgga acctgagcgt gaagatcaga gaagccaccg
1440gactggcctg ggagtggtgg cggacagtgt acgaaaagac cgacctgccc
ctcgtgcgga 1500agcggaccat ctctatctgg ggcaccacgc tgtatcctca
ggtggaagat aaggtggaaa 1560acgactgata ataggctgga gcctcggtgg
ccatgcttct tgccccttgg gcctcccccc 1620agcccctcct ccccttcctg
cacccgtacc cccgtggtct ttgaataaag tctgagtggg 1680cggc
168451649RNAS.pneumoniae 5uuacaagacc aaccuugauu gacuuagaua
agguauuuau guuggauaau acgguuauuc 60cgacuucuua ucuagccaga cggcgacgca
augucucaga agaauuguac gaggaaauuu 120uggaucacuu aguccaacca
cggcugauuu cgcugaacaa gucugaguuu augcaacuca 180auccaggaac
uuauuaggag gagaagaugg caaauaaagc aguaaaugac uuuauacuag
240cuaugaauua cgauaaaaag aaacucuuga cccaucaggg agaaaguauu
gaaaaucguu 300ucaucaaaga ggguaaucag cuacccgaug aguuuguugu
uaucgaaaga aagaagcgga 360gcuugucgac aaauacaagu gauauuucug
uaacagcuac caacgacagu cgccucuauc 420cuggagcacu ucucguagug
gaugagaccu uguuagagaa uaaucccacu cuucuugcgg 480uugaucgugc
uccgaugacu uauaguauug auuugccugg uuuggcaagu agcgauagcu
540uucuccaagu ggaagacccc agcaauucaa guguucgcgg agcgguaaac
gauuuguugg 600cuaaguggca ucaagauuau ggucagguca auaauguccc
agcuagaaug caguaugaaa 660aaauaacggc ucacagcaug gaacaacuca
aggucaaguu ugguucugac uuugaaaaga 720cagggaauuc ucuugauauu
gauuuuaacu cuguccauuc aggugaaaag cagauucaga 780uuguuaauuu
uaagcagauu uauuauacag ucagcguaga cgcuguuaaa aauccaggag
840auguguuuca agauacugua acgguagagg auuuaaaaca gagaggaauu
ucugcagagc 900guccuuuggu cuauauuucg aguguugcuu augggcgcca
agucuaucuc aaguuggaaa 960ccacgaguaa gagugaugaa guagaggcug
cuuuugaagc uuugauaaaa ggagucaagg 1020uagcuccuca gacagagugg
aagcagauuu uggacaauac agaagugaag gcgguuauuu 1080uagggggcga
cccaaguucg ggugcccgag uuguaacagg caagguggau augguagagg
1140acuugauuca agaaggcagu cgcuuuacag cagaucaucc aggcuugccg
auuuccuaua 1200caacuucuuu uuuacgugac aauguaguug cgaccuuuca
aaacaguaca gacuauguug 1260agacuaaggu uacagcuuac agaaacggag
auuuacugcu ggaucauagu ggugccuaug 1320uugcccaaua uuauauuacu
ugggaugaau uauccuauga ucaucaaggu aaggaagucu 1380ugacuccuaa
ggcuugggac agaaaugggc aggauuugac ggcucacuuu accacuagua
1440uuccuuuaaa agggaauguu cguaaucucu cugucaaaau uagagagugu
accgggcuug 1500ccugggaaug guggcguacg guuuaugaaa aaaccgauuu
gccacuagug cguaagcgga 1560cgauuucuau uuggggaaca acucucuauc
cucagguaga ggauaaggua gaaaaugacu 1620aggagaggag aaugcuugcg
acaaaaaga 164961684RNAArtificial SequenceSynthetic polynucleotide
6ucaagcuuuu ggacccucgu acagaagcua auacgacuca cuauagggaa auaagagaga
60aaagaagagu aagaagaaau auaagagcca ccauggaaac cccugcccag cugcuguucc
120ugcugcugcu guggcugccu gacaccaccg gcauggccaa caaggccgug
aacgacuuca 180uccuggccau gaacuacgac aagaagaagc ugcugaccca
ccagggcgag agcaucgaga 240acagauucau caaagagggc aaccagcugc
ccgacgaguu cgucgugauc gagcggaaga 300agcggagccu gagcaccgac
accagcgaca ucagcgugac cgccaccaac gacgccagac 360uguauccugg
cgcucugcug gugguggacg agacacugcu ggaaaacaac cccauccugc
420uggccgugga cagagccccc augaccuaca gcaucgaccu gccuggccug
gccagcagcg 480auagcuuucu gcagguggaa gaucccagca acagcgccgu
gcggggagcc gugaaugacc 540ugcuggcuaa guggcaccag gacuacggcc
aagugaacaa cgugcccgcc agaaugcagu 600acgagaagau caccgcccac
uccauggaac agcugaaagu gaaguucggc agcgacuucg 660agaaaaccgg
caacagccug gacaucgacu ucaacagcgu gcacagcggc gagaagcaga
720uccagaucgu gaacuucaag cagaucuacu acaccguguc cgugcgggcc
gugaagaacc 780cuggggacgu guuccaggau accgugaccg uggaagaucu
gaagcagcgg ggcaucagcg 840ccgagaggcc acugguguac aucagcagcg
uggccuacgg cagacaggug uaccugaagc 900uggaaaccac cuccaagagc
gacgaggugg aagccgccuu cgaggcccug aucaagggcg 960ugaaaguggc
cccucagacc gaguggaagc agauucugga caacaccgaa gugaaagccg
1020ugauccuggg cggcgacccu ucuagcggag ccagagucgu gacaggcaag
guggacaugg 1080uggaagaucu gauccaggaa ggcagccggu ucaccgccga
ucacccuggc cugccuauca 1140gcuacaccac aagcuuucug agagacaacg
ugguggccac auuccagaac uccgccgacu 1200acguggaaac aaaagugacc
gccuaccgga acggcgaucu gcugcuggau cacuccggcg 1260ccuauguggc
ccaguacuac aucaccuggg acgagcugag cuacgaucac cagggcaaag
1320aggugcugac ccccaaggcc ugggacagaa acggccagga ucugacagcc
cacuucacaa 1380ccagcauccc ccugaagggc aacgugcgga accugagcgu
gaagaucaga gagugcaccg 1440gacuggccug ggaguggugg cggaccgugu
acgaaaagac cgaccugccc cucgugcgga 1500agcggaccau cucuaucugg
ggcaccacgc uguauccuca gguggaagau aagguggaaa 1560acgacugaua
auaggcugga gccucggugg ccaugcuucu ugccccuugg gccucccccc
1620agccccuccu ccccuuccug cacccguacc cccguggucu uugaauaaag
ucugaguggg 1680cggc 168471684RNAArtificial SequenceSynthetic
polynucleotide 7ucaagcuuuu ggacccucgu acagaagcua auacgacuca
cuauagggaa auaagagaga 60aaagaagagu aagaagaaau auaagagcca ccauggaaac
cccugcccag cugcuguucc 120ugcugcugcu guggcugccu gacaccaccg
gcauggccaa caaggccgug aacgacuuca 180uccuggccau gaacuacgac
aagaagaagc ugcugaccca ccagggcgag agcaucgaga 240acagauucau
caaagagggc aaccagcugc ccgacgaguu cgucgugauc gagcggaaga
300agcggagccu gagcaccgac accagcgaca ucagcgugac cgccaccaac
gacgccagac 360uguauccugg cgcucugcug gugguggacg agacacugcu
ggaaaacaac cccauccugc 420uggccgugga cagagccccc augaccuaca
gcaucgaccu gccuggccug gccagcagcg 480auagcuuucu gcagguggaa
gaucccagca acagcgccgu gcggggagcc gugaaugacc 540ugcuggcuaa
guggcaccag gacuacggcc aagugaacaa cgugcccgcc agaaugcagu
600acgagaagau caccgcccac uccauggaac agcugaaagu gaaguucggc
agcgacuucg 660agaaaaccgg caacagccug gacaucgacu ucaacagcgu
gcacagcggc gagaagcaga 720uccagaucgu gaacuucaag cagaucuacu
acaccguguc cguggacgcc gugaagaacc 780ccggggacgu guuccaggau
accgugaccg uggaagaucu gaagcagcgg ggcaucagcg 840ccgagaggcc
acugguguac aucagcagcg uggccuacgg cagacaggug uaccugaagc
900uggaaaccac cuccaagagc gacgaggugg aagccgccuu cgaggcccug
aucaagggcg 960ugaaaguggc cccucagacc gaguggaagc agauucugga
caacaccgaa gugaaagccg 1020ugauccuggg cggcgacccu ucuagcggag
ccagagucgu gacaggcaag guggacaugg 1080uggaagaucu gauccaggaa
ggcagccggu ucaccgccga ucacccuggc cugccuauca 1140gcuacaccac
aagcuuucug agagacaacg ugguggccac auuccagaac uccgccgacu
1200acguggaaac aaaagugacc gccuaccgga acggcgaucu gcugcuggau
cacuccggcg 1260ccuauguggc ccaguacuac aucaccuggg acgagcugag
cuacgaucac cagggcaaag 1320aggugcugac ccccaaggcc ugggacagaa
acggccagga ucugacagcc cacuucacaa 1380ccagcauccc ccugaagggc
aacgugcgga accugagcgu gaagaucaga gagugcaccg 1440gacuggccug
ggaguggugg cggaccgugu acgaaaagac cgaccugccc cucgugcgga
1500agcggaccau cucuaucugg ggcaccaccg acuaccccca gguggaagau
aagguggaaa 1560acgacugaua auaggcugga gccucggugg ccaugcuucu
ugccccuugg gccucccccc 1620agccccuccu ccccuuccug cacccguacc
cccguggucu uugaauaaag ucugaguggg 1680cggc 168481684RNAArtificial
SequenceSynthetic polynucleotide 8ucaagcuuuu ggacccucgu acagaagcua
auacgacuca cuauagggaa auaagagaga 60aaagaagagu aagaagaaau auaagagcca
ccauggaaac cccugcccag cugcuguucc 120ugcugcugcu guggcugccu
gacaccaccg gcauggccaa caaggccgug aacgacuuca 180uccuggccau
gaacuacgac aagaagaagc ugcugaccca ccagggcgag agcaucgaga
240acagauucau caaagagggc aaccagcugc ccgacgaguu cgucgugauc
gagcggaaga 300agcggagccu gagcaccgac accagcgaca ucagcgugac
cgccugcaac gacgccagac 360uguauccugg cgcucugcug gugguggacg
agacacugcu ggaaaacaac cccauccugc 420uggccgugga cagagccccc
augaccuaca gcaucgaccu gccuggccug gccagcagcg 480auagcuuucu
gcagguggaa gaucccagca acagcgccgu gcggggagcc gugaaugacc
540ugcuggcuaa guggcaccag gacuacggcc aagugaacaa cgugcccgcc
agaaugcagu 600acgagaagau caccgcccac uccauggaac agcugaaagu
gaaguucggc agcgacuucg 660agaaaaccgg caacagccug gacaucgacu
ucaacagcgu gcacagcggc gagaagcaga 720uccagaucgu gaacuucaag
cagaucuacu acaccguguc cguggacgcc gugaagaacc 780ccggggacgu
guuccaggau accgugaccg uggaagaucu gaagcagcgg ggcaucagcg
840ccgagaggcc acugguguac aucagcagcg uggccuacgg cagacaggug
uaccugaagc 900uggaaaccac cuccaagagc gacgaggugg aagccgccuu
cgaggcccug aucaagggcg 960ugaaaguggc cccucagacc gaguggaagc
agauucugga caacaccgaa gugaaagccg 1020ugauccugug cggcgacccu
ucuagcggag ccagagucgu gacaggcaag guggacaugg 1080uggaagaucu
gauccaggaa ggcagccggu ucaccgccga ucacccuggc cugccuauca
1140gcuacaccac aagcuuucug agagacaacg ugguggccac auuccagaac
uccgccgacu 1200acguggaaac aaaagugaca gccuaccgga acggcgaucu
gcugcuggau cacuccggcg 1260ccuauguggc ccaguacuac aucaccuggg
acgagcugag cuacgaucac cagggcaaag 1320aggugcugac ccccaaggcc
ugggacagaa acggccagga ucugacagcc cacuucacaa 1380ccagcauccc
ccugaagggc aacgugcgga accugagcgu gaagaucaga gaagccaccg
1440gacuggccug ggaguggugg cggacagugu acgaaaagac cgaccugccc
cucgugcgga 1500agcggaccau cucuaucugg ggcaccacgc uguauccuca
gguggaagau aagguggaaa 1560acgacugaua auaggcugga gccucggugg
ccaugcuucu ugccccuugg gccucccccc 1620agccccuccu ccccuuccug
cacccguacc cccguggucu uugaauaaag ucugaguggg 1680cggc
16849471PRTS.pneumoniae 9Met Ala Asn Lys Ala Val Asn Asp Phe Ile
Leu Ala Met Asn Tyr Asp1 5 10 15Lys Lys Lys Leu Leu Thr His Gln Gly
Glu Ser Ile Glu Asn Arg Phe 20 25 30Ile Lys Glu Gly Asn Gln Leu Pro
Asp Glu Phe Val Val Ile Glu Arg 35 40 45Lys Lys Arg Ser Leu Ser Thr
Asn Thr Ser Asp Ile Ser Val Thr Ala 50 55 60Thr Asn Asp Ser Arg Leu
Tyr Pro Gly Ala Leu Leu Val Val Asp Glu65 70 75 80Thr Leu Leu Glu
Asn Asn Pro Thr Leu Leu Ala Val Asp Arg Ala Pro 85 90 95Met Thr Tyr
Ser Ile Asp Leu Pro Gly Leu Ala Ser Ser Asp Ser Phe 100 105 110Leu
Gln Val Glu Asp Pro Ser Asn Ser Ser Val Arg Gly Ala Val Asn 115 120
125Asp Leu Leu Ala Lys Trp His Gln Asp Tyr Gly Gln Val Asn Asn Val
130 135 140Pro Ala Arg Met Gln
Tyr Glu Lys Ile Thr Ala His Ser Met Glu Gln145 150 155 160Leu Lys
Val Lys Phe Gly Ser Asp Phe Glu Lys Thr Gly Asn Ser Leu 165 170
175Asp Ile Asp Phe Asn Ser Val His Ser Gly Glu Lys Gln Ile Gln Ile
180 185 190Val Asn Phe Lys Gln Ile Tyr Tyr Thr Val Ser Val Asp Ala
Val Lys 195 200 205Asn Pro Gly Asp Val Phe Gln Asp Thr Val Thr Val
Glu Asp Leu Lys 210 215 220Gln Arg Gly Ile Ser Ala Glu Arg Pro Leu
Val Tyr Ile Ser Ser Val225 230 235 240Ala Tyr Gly Arg Gln Val Tyr
Leu Lys Leu Glu Thr Thr Ser Lys Ser 245 250 255Asp Glu Val Glu Ala
Ala Phe Glu Ala Leu Ile Lys Gly Val Lys Val 260 265 270Ala Pro Gln
Thr Glu Trp Lys Gln Ile Leu Asp Asn Thr Glu Val Lys 275 280 285Ala
Val Ile Leu Gly Gly Asp Pro Ser Ser Gly Ala Arg Val Val Thr 290 295
300Gly Lys Val Asp Met Val Glu Asp Leu Ile Gln Glu Gly Ser Arg
Phe305 310 315 320Thr Ala Asp His Pro Gly Leu Pro Ile Ser Tyr Thr
Thr Ser Phe Leu 325 330 335Arg Asp Asn Val Val Ala Thr Phe Gln Asn
Ser Thr Asp Tyr Val Glu 340 345 350Thr Lys Val Thr Ala Tyr Arg Asn
Gly Asp Leu Leu Leu Asp His Ser 355 360 365Gly Ala Tyr Val Ala Gln
Tyr Tyr Ile Thr Trp Asn Glu Leu Ser Tyr 370 375 380Asp His Gln Gly
Lys Glu Val Leu Thr Pro Lys Ala Trp Asp Arg Asn385 390 395 400Gly
Gln Asp Leu Thr Ala His Phe Thr Thr Ser Ile Pro Leu Lys Gly 405 410
415Asn Val Arg Asn Leu Ser Val Lys Ile Arg Glu Cys Thr Gly Leu Ala
420 425 430Trp Glu Trp Trp Arg Thr Val Tyr Glu Lys Thr Asp Leu Pro
Leu Val 435 440 445Arg Lys Arg Thr Ile Ser Ile Trp Gly Thr Thr Leu
Tyr Pro Gln Val 450 455 460Glu Asp Lys Val Glu Asn Asp465
47010477PRTArtificial SequenceSynthetic polypeptide 10His His His
His His His Gly Ser Ala Asn Lys Ala Val Asn Asp Phe1 5 10 15Ile Leu
Ala Met Asn Tyr Asp Lys Lys Lys Leu Leu Thr His Gln Gly 20 25 30Glu
Ser Ile Glu Asn Arg Phe Ile Lys Glu Gly Asn Gln Leu Pro Asp 35 40
45Glu Phe Val Val Ile Glu Arg Lys Lys Arg Ser Leu Ser Thr Ser Thr
50 55 60Ser Asp Ile Ser Val Thr Ala Thr Asn Asp Ser Arg Leu Tyr Pro
Gly65 70 75 80Ala Leu Leu Val Val Asp Glu Thr Leu Leu Glu Asn Asn
Pro Thr Leu 85 90 95Leu Ala Val Asp Arg Ala Pro Met Thr Tyr Ser Ile
Asp Leu Pro Gly 100 105 110Leu Ala Ser Ser Asp Ser Phe Leu Gln Val
Glu Asp Pro Ser Asn Ser 115 120 125Ser Val Arg Gly Ala Val Asn Asp
Leu Leu Ala Lys Trp His Gln Asp 130 135 140Tyr Gly Gln Val Asn Asn
Val Pro Ala Arg Met Gln Tyr Glu Lys Ile145 150 155 160Thr Ala His
Ser Met Glu Gln Leu Lys Val Lys Phe Gly Ser Asp Phe 165 170 175Glu
Lys Thr Gly Asn Ser Leu Asp Ile Asp Phe Asn Ser Val His Ser 180 185
190Gly Glu Lys Gln Ile Gln Ile Val Asn Phe Lys Gln Ile Tyr Tyr Thr
195 200 205Val Ser Val Asp Ala Val Arg Asn Pro Gly Asp Val Phe Gln
Asp Thr 210 215 220Val Thr Val Glu Asp Leu Lys Gln Arg Gly Ile Ser
Ala Glu Arg Pro225 230 235 240Leu Val Tyr Ile Ser Ser Val Ala Tyr
Gly Arg Gln Val Tyr Leu Lys 245 250 255Leu Glu Thr Thr Ser Lys Ser
Asp Glu Val Glu Ala Ala Phe Glu Ala 260 265 270Leu Ile Lys Gly Val
Lys Val Ala Pro Gln Thr Glu Trp Lys Gln Ile 275 280 285Leu Asp Asn
Thr Glu Val Lys Ala Val Ile Leu Gly Gly Asp Pro Ser 290 295 300Ser
Gly Ala Arg Val Val Thr Gly Lys Val Asp Met Val Asp Asp Leu305 310
315 320Ile Gln Glu Gly Ser Arg Phe Thr Ala Asp His Pro Gly Leu Pro
Ile 325 330 335Ser Tyr Thr Thr Ser Phe Leu Arg Asp Asn Val Val Ala
Thr Phe Gln 340 345 350Asn Ser Thr Asp Tyr Val Glu Thr Lys Val Thr
Ala Tyr Arg Asn Gly 355 360 365Asp Leu Leu Leu Asp His Ser Gly Ala
Tyr Val Ala Gln Tyr Tyr Ile 370 375 380Thr Trp Asp Glu Leu Ser Tyr
Asp His Gln Gly Lys Glu Val Leu Thr385 390 395 400Pro Lys Ala Trp
Asp Arg Asn Gly Gln Asp Leu Thr Ala His Phe Thr 405 410 415Thr Ser
Ile Pro Leu Lys Gly Asn Val Arg Asn Leu Ser Val Lys Ile 420 425
430Arg Glu Cys Thr Gly Leu Ala Trp Glu Trp Trp Arg Thr Val Tyr Glu
435 440 445Lys Thr Asp Leu Pro Leu Val Arg Lys Arg Thr Ile Ser Ile
Trp Gly 450 455 460Thr Thr Leu Tyr Pro Gln Val Glu Asp Lys Val Glu
Asn465 470 47511477PRTArtificial SequenceSynthetic polypeptide
11His His His His His His Gly Ser Ala Asn Lys Ala Val Asn Asp Phe1
5 10 15Ile Leu Ala Met Asn Tyr Asp Lys Lys Lys Leu Leu Thr His Gln
Gly 20 25 30Glu Ser Ile Glu Asn Arg Phe Ile Lys Glu Gly Asn Gln Leu
Pro Asp 35 40 45Glu Phe Val Val Ile Glu Arg Lys Lys Arg Ser Leu Ser
Thr Ser Thr 50 55 60Ser Asp Ile Ser Val Thr Ala Thr Asn Asp Ser Arg
Leu Tyr Pro Gly65 70 75 80Ala Leu Leu Val Val Asp Glu Thr Leu Leu
Glu Asn Asn Pro Thr Leu 85 90 95Leu Ala Val Asp Arg Ala Pro Met Thr
Tyr Ser Ile Phe Leu Pro Gly 100 105 110Leu Ala Ser Ser Asp Ser Phe
Leu Gln Val Glu Asp Pro Ser Asn Ser 115 120 125Ser Val Arg Gly Ala
Val Asn Asp Leu Leu Ala Lys Trp His Gln Asp 130 135 140Tyr Gly Gln
Val Asn Asn Val Pro Ala Arg Met Gln Tyr Glu Lys Ile145 150 155
160Thr Ala His Ser Met Glu Gln Leu Lys Val Lys Phe Gly Ser Asp Phe
165 170 175Glu Lys Thr Gly Asn Ser Leu Asp Ile Asp Phe Asn Ser Val
His Ser 180 185 190Gly Glu Lys Gln Ile Gln Ile Val Asn Phe Lys Gln
Ile Tyr Tyr Thr 195 200 205Val Ser Val Asp Ala Val Arg Asn Pro Gly
Asp Val Phe Gln Asp Thr 210 215 220Val Thr Val Glu Asp Leu Lys Gln
Arg Gly Ile Ser Ala Glu Arg Pro225 230 235 240Leu Val Tyr Ile Ser
Ser Val Ala Tyr Gly Arg Gln Val Tyr Leu Lys 245 250 255Leu Glu Thr
Thr Ser Lys Ser Asp Glu Val Glu Ala Ala Phe Glu Ala 260 265 270Leu
Ile Lys Gly Val Lys Val Ala Pro Gln Thr Glu Trp Lys Gln Ile 275 280
285Leu Asp Asn Thr Glu Val Lys Ala Val Ile Leu Gly Gly Asp Pro Ser
290 295 300Ser Gly Ala Arg Val Val Thr Gly Lys Val Asp Met Val Asp
Asp Leu305 310 315 320Ile Gln Glu Gly Ser Arg Phe Thr Ala Asp His
Pro Gly Leu Pro Ile 325 330 335Ser Tyr Thr Thr Ser Phe Leu Arg Asp
Asn Val Val Ala Thr Phe Gln 340 345 350Asn Ser Thr Asp Tyr Val Glu
Thr Lys Val Thr Ala Tyr Arg Asn Gly 355 360 365Asp Leu Leu Leu Asp
His Ser Gly Ala Tyr Val Ala Gln Tyr Tyr Ile 370 375 380Thr Trp Asp
Glu Leu Ser Tyr Asp His Gln Gly Lys Glu Val Leu Thr385 390 395
400Pro Lys Ala Trp Asp Arg Asn Gly Gln Asp Leu Thr Ala His Phe Thr
405 410 415Thr Ser Ile Pro Leu Lys Gly Asn Val Arg Asn Leu Ser Val
Lys Ile 420 425 430Arg Glu Cys Thr Gly Leu Ala Trp Glu Trp Trp Arg
Thr Val Tyr Glu 435 440 445Lys Thr Asp Leu Pro Leu Val Arg Lys Arg
Thr Ile Ser Ile Trp Gly 450 455 460Thr Thr Leu Tyr Pro Gln Val Glu
Asp Lys Val Glu Asn465 470 47512477PRTArtificial SequenceSynthetic
polypeptide 12His His His His His His Gly Ser Ala Asn Lys Ala Val
Asn Asp Phe1 5 10 15Ile Leu Ala Met Asn Tyr Asp Lys Lys Lys Leu Leu
Thr His Gln Gly 20 25 30Glu Ser Ile Glu Asn Arg Phe Ile Lys Glu Gly
Asn Gln Leu Pro Asp 35 40 45Glu Phe Val Val Ile Glu Arg Lys Lys Arg
Ser Leu Ser Thr Ser Thr 50 55 60Ser Asp Ile Ser Val Thr Ala Thr Asn
Asp Ser Arg Leu Tyr Pro Gly65 70 75 80Ala Leu Leu Val Val Asp Glu
Thr Leu Leu Glu Asn Asn Pro Thr Leu 85 90 95Leu Ala Val Asp Arg Ala
Pro Met Thr Tyr Ser Ile Met Leu Pro Gly 100 105 110Leu Ala Ser Ser
Asp Ser Phe Leu Gln Val Glu Asp Pro Ser Asn Ser 115 120 125Ser Val
Arg Gly Ala Val Asn Asp Leu Leu Ala Lys Trp His Gln Asp 130 135
140Tyr Gly Gln Val Asn Asn Val Pro Ala Arg Met Gln Tyr Glu Lys
Ile145 150 155 160Thr Ala His Ser Met Glu Gln Leu Lys Val Lys Phe
Gly Ser Asp Phe 165 170 175Glu Lys Thr Gly Asn Ser Leu Asp Ile Asp
Phe Asn Ser Val His Ser 180 185 190Gly Glu Lys Gln Ile Gln Ile Val
Asn Phe Lys Gln Ile Tyr Tyr Thr 195 200 205Val Ser Val Asp Ala Val
Arg Asn Pro Gly Asp Val Phe Gln Asp Thr 210 215 220Val Thr Val Glu
Asp Leu Lys Gln Arg Gly Ile Ser Ala Glu Arg Pro225 230 235 240Leu
Val Tyr Ile Ser Ser Val Ala Tyr Gly Arg Gln Val Tyr Leu Lys 245 250
255Leu Glu Thr Thr Ser Lys Ser Asp Glu Val Glu Ala Ala Phe Glu Ala
260 265 270Leu Ile Lys Gly Val Lys Val Ala Pro Gln Thr Glu Trp Lys
Gln Ile 275 280 285Leu Asp Asn Thr Glu Val Lys Ala Val Ile Leu Gly
Gly Asp Pro Ser 290 295 300Ser Gly Ala Arg Val Val Thr Gly Lys Val
Asp Met Val Asp Asp Leu305 310 315 320Ile Gln Glu Gly Ser Arg Phe
Thr Ala Asp His Pro Gly Leu Pro Ile 325 330 335Ser Tyr Thr Thr Ser
Phe Leu Arg Asp Asn Val Val Ala Thr Phe Gln 340 345 350Asn Ser Thr
Asp Tyr Val Glu Thr Lys Val Thr Ala Tyr Arg Asn Gly 355 360 365Asp
Leu Leu Leu Asp His Ser Gly Ala Tyr Val Ala Gln Tyr Tyr Ile 370 375
380Thr Trp Asp Glu Leu Ser Tyr Asp His Gln Gly Lys Glu Val Leu
Thr385 390 395 400Pro Lys Ala Trp Asp Arg Asn Gly Gln Asp Leu Thr
Ala His Phe Thr 405 410 415Thr Ser Ile Pro Leu Lys Gly Asn Val Arg
Asn Leu Ser Val Lys Ile 420 425 430Arg Glu Cys Thr Gly Leu Ala Trp
Glu Trp Trp Arg Thr Val Tyr Glu 435 440 445Lys Thr Asp Leu Pro Leu
Val Arg Lys Arg Thr Ile Ser Ile Trp Gly 450 455 460Thr Thr Leu Tyr
Pro Gln Val Glu Asp Lys Val Glu Asn465 470 47513477PRTArtificial
SequenceSynthetic polypeptide 13His His His His His His Gly Ser Ala
Asn Lys Ala Val Asn Asp Phe1 5 10 15Ile Leu Ala Met Asn Tyr Asp Lys
Lys Lys Leu Leu Thr His Gln Gly 20 25 30Glu Ser Ile Glu Asn Arg Phe
Ile Lys Glu Gly Asn Gln Leu Pro Asp 35 40 45Glu Phe Val Val Ile Glu
Arg Lys Lys Arg Ser Leu Ser Thr Ser Thr 50 55 60Ser Asp Ile Ser Val
Thr Ala Thr Asn Asp Ser Arg Leu Tyr Pro Gly65 70 75 80Ala Leu Leu
Val Val Asp Glu Thr Leu Leu Glu Asn Asn Pro Thr Leu 85 90 95Leu Ala
Val Asp Arg Ala Pro Met Thr Tyr Ser Ile Asp Leu Pro Gly 100 105
110Leu Met Ser Ser Asp Ser Phe Leu Gln Val Glu Asp Pro Ser Asn Ser
115 120 125Ser Val Arg Gly Ala Val Asn Asp Leu Leu Ala Lys Trp His
Gln Asp 130 135 140Tyr Gly Gln Val Asn Asn Val Pro Ala Arg Met Gln
Tyr Glu Lys Ile145 150 155 160Thr Ala His Ser Met Glu Gln Leu Lys
Val Lys Phe Gly Ser Asp Phe 165 170 175Glu Lys Thr Gly Asn Ser Leu
Asp Ile Asp Phe Asn Ser Val His Ser 180 185 190Gly Glu Lys Gln Ile
Gln Ile Val Asn Phe Lys Gln Ile Tyr Tyr Thr 195 200 205Val Ser Val
Asp Ala Val Arg Asn Pro Gly Asp Val Phe Gln Asp Thr 210 215 220Val
Thr Val Glu Asp Leu Lys Gln Arg Gly Ile Ser Ala Glu Arg Pro225 230
235 240Leu Val Tyr Ile Ser Ser Val Ala Tyr Gly Arg Gln Val Tyr Leu
Lys 245 250 255Leu Glu Thr Thr Ser Lys Ser Asp Glu Val Glu Ala Ala
Phe Glu Ala 260 265 270Leu Ile Lys Gly Val Lys Val Ala Pro Gln Thr
Glu Trp Lys Gln Ile 275 280 285Leu Asp Asn Thr Glu Val Lys Ala Val
Ile Leu Gly Gly Asp Pro Ser 290 295 300Ser Gly Ala Arg Val Val Thr
Gly Lys Val Asp Met Val Asp Asp Leu305 310 315 320Ile Gln Glu Gly
Ser Arg Phe Thr Ala Asp His Pro Gly Leu Pro Ile 325 330 335Ser Tyr
Thr Thr Ser Phe Leu Arg Asp Asn Val Val Ala Thr Phe Gln 340 345
350Asn Ser Thr Asp Tyr Val Glu Thr Lys Val Thr Ala Tyr Arg Asn Gly
355 360 365Asp Leu Leu Leu Asp His Ser Gly Ala Tyr Val Ala Gln Tyr
Tyr Ile 370 375 380Thr Trp Asp Glu Leu Ser Tyr Asp His Gln Gly Lys
Glu Val Leu Thr385 390 395 400Pro Lys Ala Trp Asp Arg Asn Gly Gln
Asp Leu Thr Ala His Phe Thr 405 410 415Thr Ser Ile Pro Leu Lys Gly
Asn Val Arg Asn Leu Ser Val Lys Ile 420 425 430Arg Glu Cys Thr Gly
Leu Ala Trp Glu Trp Trp Arg Thr Val Tyr Glu 435 440 445Lys Thr Asp
Leu Pro Leu Val Arg Lys Arg Thr Ile Ser Ile Trp Gly 450 455 460Thr
Thr Leu Tyr Pro Gln Val Glu Asp Lys Val Glu Asn465 470
47514477PRTArtificial SequenceSynthetic polypeptide 14His His His
His His His Gly Ser Ala Asn Lys Ala Val Asn Asp Phe1 5 10 15Ile Leu
Ala Met Asn Tyr Asp Lys Lys Lys Leu Leu Thr His Gln Gly 20 25 30Glu
Ser Ile Glu Asn Arg Phe Ile Lys Glu Gly Asn Gln Leu Pro Asp 35 40
45Glu Phe Val Val Ile Glu Arg Lys Lys Arg Ser Leu Ser Thr Ser Thr
50 55 60Ser Asp Ile Ser Val Thr Ala Thr Asn Asp Ser Arg Leu Tyr Pro
Gly65 70 75 80Ala Leu Leu Val Val Asp Glu Thr Leu Leu Glu Asn Asn
Pro Thr Leu 85 90 95Leu Ala Val Asp Arg Ala Pro Met Thr Tyr Ser Ile
Asp Leu Pro Gly 100 105 110Leu Gln Ser Ser Asp Ser Phe Leu Gln Val
Glu Asp Pro Ser Asn Ser 115 120 125Ser Val Arg Gly Ala Val Asn Asp
Leu Leu Ala Lys Trp His Gln Asp 130 135 140Tyr Gly Gln Val Asn Asn
Val Pro Ala Arg Met Gln Tyr Glu Lys Ile145 150 155 160Thr Ala His
Ser Met Glu Gln Leu Lys Val Lys Phe Gly Ser Asp Phe 165 170 175Glu
Lys Thr Gly Asn Ser Leu Asp Ile Asp Phe Asn Ser Val His Ser 180 185
190Gly Glu Lys Gln Ile Gln Ile Val Asn Phe Lys Gln Ile Tyr Tyr Thr
195 200 205Val Ser Val Asp
Ala Val Arg Asn Pro Gly Asp Val Phe Gln Asp Thr 210 215 220Val Thr
Val Glu Asp Leu Lys Gln Arg Gly Ile Ser Ala Glu Arg Pro225 230 235
240Leu Val Tyr Ile Ser Ser Val Ala Tyr Gly Arg Gln Val Tyr Leu Lys
245 250 255Leu Glu Thr Thr Ser Lys Ser Asp Glu Val Glu Ala Ala Phe
Glu Ala 260 265 270Leu Ile Lys Gly Val Lys Val Ala Pro Gln Thr Glu
Trp Lys Gln Ile 275 280 285Leu Asp Asn Thr Glu Val Lys Ala Val Ile
Leu Gly Gly Asp Pro Ser 290 295 300Ser Gly Ala Arg Val Val Thr Gly
Lys Val Asp Met Val Asp Asp Leu305 310 315 320Ile Gln Glu Gly Ser
Arg Phe Thr Ala Asp His Pro Gly Leu Pro Ile 325 330 335Ser Tyr Thr
Thr Ser Phe Leu Arg Asp Asn Val Val Ala Thr Phe Gln 340 345 350Asn
Ser Thr Asp Tyr Val Glu Thr Lys Val Thr Ala Tyr Arg Asn Gly 355 360
365Asp Leu Leu Leu Asp His Ser Gly Ala Tyr Val Ala Gln Tyr Tyr Ile
370 375 380Thr Trp Asp Glu Leu Ser Tyr Asp His Gln Gly Lys Glu Val
Leu Thr385 390 395 400Pro Lys Ala Trp Asp Arg Asn Gly Gln Asp Leu
Thr Ala His Phe Thr 405 410 415Thr Ser Ile Pro Leu Lys Gly Asn Val
Arg Asn Leu Ser Val Lys Ile 420 425 430Arg Glu Cys Thr Gly Leu Ala
Trp Glu Trp Trp Arg Thr Val Tyr Glu 435 440 445Lys Thr Asp Leu Pro
Leu Val Arg Lys Arg Thr Ile Ser Ile Trp Gly 450 455 460Thr Thr Leu
Tyr Pro Gln Val Glu Asp Lys Val Glu Asn465 470
47515477PRTArtificial SequenceSynthetic polypeptide 15His His His
His His His Gly Ser Ala Asn Lys Ala Val Asn Asp Phe1 5 10 15Ile Leu
Ala Met Asn Tyr Asp Lys Lys Lys Leu Leu Thr His Gln Gly 20 25 30Glu
Ser Ile Glu Asn Arg Phe Ile Lys Glu Gly Asn Gln Leu Pro Asp 35 40
45Glu Phe Val Val Ile Glu Arg Lys Lys Arg Ser Leu Ser Thr Ser Thr
50 55 60Ser Asp Ile Ser Val Thr Ala Thr Asn Asp Ser Arg Leu Tyr Pro
Gly65 70 75 80Ala Leu Leu Val Val Asp Glu Thr Leu Leu Glu Asn Asn
Pro Thr Leu 85 90 95Leu Ala Val Asp Arg Ala Pro Met Thr Tyr Ser Ile
Asp Leu Pro Gly 100 105 110Leu Ala Ser Ser Asp Ser Phe Leu Gln Val
Glu Asp Pro Ser Phe Ser 115 120 125Ser Val Arg Gly Ala Val Asn Asp
Leu Leu Ala Lys Trp His Gln Asp 130 135 140Tyr Gly Gln Val Asn Asn
Val Pro Ala Arg Met Gln Tyr Glu Lys Ile145 150 155 160Thr Ala His
Ser Met Glu Gln Leu Lys Val Lys Phe Gly Ser Asp Phe 165 170 175Glu
Lys Thr Gly Asn Ser Leu Asp Ile Asp Phe Asn Ser Val His Ser 180 185
190Gly Glu Lys Gln Ile Gln Ile Val Asn Phe Lys Gln Ile Tyr Tyr Thr
195 200 205Val Ser Val Asp Ala Val Arg Asn Pro Gly Asp Val Phe Gln
Asp Thr 210 215 220Val Thr Val Glu Asp Leu Lys Gln Arg Gly Ile Ser
Ala Glu Arg Pro225 230 235 240Leu Val Tyr Ile Ser Ser Val Ala Tyr
Gly Arg Gln Val Tyr Leu Lys 245 250 255Leu Glu Thr Thr Ser Lys Ser
Asp Glu Val Glu Ala Ala Phe Glu Ala 260 265 270Leu Ile Lys Gly Val
Lys Val Ala Pro Gln Thr Glu Trp Lys Gln Ile 275 280 285Leu Asp Asn
Thr Glu Val Lys Ala Val Ile Leu Gly Gly Asp Pro Ser 290 295 300Ser
Gly Ala Arg Val Val Thr Gly Lys Val Asp Met Val Asp Asp Leu305 310
315 320Ile Gln Glu Gly Ser Arg Phe Thr Ala Asp His Pro Gly Leu Pro
Ile 325 330 335Ser Tyr Thr Thr Ser Phe Leu Arg Asp Asn Val Val Ala
Thr Phe Gln 340 345 350Asn Ser Thr Asp Tyr Val Glu Thr Lys Val Thr
Ala Tyr Arg Asn Gly 355 360 365Asp Leu Leu Leu Asp His Ser Gly Ala
Tyr Val Ala Gln Tyr Tyr Ile 370 375 380Thr Trp Asp Glu Leu Ser Tyr
Asp His Gln Gly Lys Glu Val Leu Thr385 390 395 400Pro Lys Ala Trp
Asp Arg Asn Gly Gln Asp Leu Thr Ala His Phe Thr 405 410 415Thr Ser
Ile Pro Leu Lys Gly Asn Val Arg Asn Leu Ser Val Lys Ile 420 425
430Arg Glu Cys Thr Gly Leu Ala Trp Glu Trp Trp Arg Thr Val Tyr Glu
435 440 445Lys Thr Asp Leu Pro Leu Val Arg Lys Arg Thr Ile Ser Ile
Trp Gly 450 455 460Thr Thr Leu Tyr Pro Gln Val Glu Asp Lys Val Glu
Asn465 470 47516477PRTArtificial SequenceSynthetic polypeptide
16His His His His His His Gly Ser Ala Asn Lys Ala Val Asn Asp Phe1
5 10 15Ile Leu Ala Met Asn Tyr Asp Lys Lys Lys Leu Leu Thr His Gln
Gly 20 25 30Glu Ser Ile Glu Asn Arg Phe Ile Lys Glu Gly Asn Gln Leu
Pro Asp 35 40 45Glu Phe Val Val Ile Glu Arg Lys Lys Arg Ser Leu Ser
Thr Ser Thr 50 55 60Ser Asp Ile Ser Val Thr Ala Thr Asn Asp Ser Arg
Leu Tyr Pro Gly65 70 75 80Ala Leu Leu Val Val Asp Glu Thr Leu Leu
Glu Asn Asn Pro Thr Leu 85 90 95Leu Ala Val Asp Arg Ala Pro Met Thr
Tyr Ser Ile Asp Leu Pro Gly 100 105 110Leu Ala Ser Ser Asp Ser Phe
Leu Gln Val Glu Asp Pro Ser Asn Ser 115 120 125Ser Val Arg Gly Ala
Val Asn Asp Leu Leu Ala Lys Trp His Gln Asp 130 135 140Tyr Gly Gln
Val Asn Asn Val Pro Ala Arg Met Gln Tyr Glu Lys Ile145 150 155
160Thr Ala His Ser Met Glu Gln Leu Lys Val Lys Phe Gly Ser Asp Phe
165 170 175Glu Lys Thr Gly Asn Ser Leu Asp Ile Asp Phe Asn Ser Val
His Ser 180 185 190Gly Arg Lys Gln Ile Gln Ile Val Asn Phe Lys Gln
Ile Tyr Tyr Thr 195 200 205Val Ser Val Asp Ala Val Arg Asn Pro Gly
Asp Val Phe Gln Asp Thr 210 215 220Val Thr Val Glu Asp Leu Lys Gln
Arg Gly Ile Ser Ala Glu Arg Pro225 230 235 240Leu Val Tyr Ile Ser
Ser Val Ala Tyr Gly Arg Gln Val Tyr Leu Lys 245 250 255Leu Glu Thr
Thr Ser Lys Ser Asp Glu Val Glu Ala Ala Phe Glu Ala 260 265 270Leu
Ile Lys Gly Val Lys Val Ala Pro Gln Thr Glu Trp Lys Gln Ile 275 280
285Leu Asp Asn Thr Glu Val Lys Ala Val Ile Leu Gly Gly Asp Pro Ser
290 295 300Ser Gly Ala Arg Val Val Thr Gly Lys Val Asp Met Val Asp
Asp Leu305 310 315 320Ile Gln Glu Gly Ser Arg Phe Thr Ala Asp His
Pro Gly Leu Pro Ile 325 330 335Ser Tyr Thr Thr Ser Phe Leu Arg Asp
Asn Val Val Ala Thr Phe Gln 340 345 350Asn Ser Thr Asp Tyr Val Glu
Thr Lys Val Thr Ala Tyr Arg Asn Gly 355 360 365Asp Leu Leu Leu Asp
His Ser Gly Ala Tyr Val Ala Gln Tyr Tyr Ile 370 375 380Thr Trp Asp
Glu Leu Ser Tyr Asp His Gln Gly Lys Glu Val Leu Thr385 390 395
400Pro Lys Ala Trp Asp Arg Asn Gly Gln Asp Leu Thr Ala His Phe Thr
405 410 415Thr Ser Ile Pro Leu Lys Gly Asn Val Arg Asn Leu Ser Val
Lys Ile 420 425 430Arg Glu Cys Thr Gly Leu Ala Trp Glu Trp Trp Arg
Thr Val Tyr Glu 435 440 445Lys Thr Asp Leu Pro Leu Val Arg Lys Arg
Thr Ile Ser Ile Trp Gly 450 455 460Thr Thr Leu Tyr Pro Gln Val Glu
Asp Lys Val Glu Asn465 470 47517477PRTArtificial SequenceSynthetic
polypeptide 17His His His His His His Gly Ser Ala Asn Lys Ala Val
Asn Asp Phe1 5 10 15Ile Leu Ala Met Asn Tyr Asp Lys Lys Lys Leu Leu
Thr His Gln Gly 20 25 30Glu Ser Ile Glu Asn Arg Phe Ile Lys Glu Gly
Asn Gln Leu Pro Asp 35 40 45Glu Phe Val Val Ile Glu Arg Lys Lys Arg
Ser Leu Ser Thr Ser Thr 50 55 60Ser Asp Ile Ser Val Thr Ala Thr Asn
Asp Ser Arg Leu Tyr Pro Gly65 70 75 80Ala Leu Leu Val Val Asp Glu
Thr Leu Leu Glu Asn Asn Pro Thr Leu 85 90 95Leu Ala Val Asp Arg Ala
Pro Met Thr Tyr Ser Ile Asp Leu Pro Gly 100 105 110Leu Ala Ser Ser
Asp Ser Phe Leu Gln Val Glu Asp Pro Ser Asn Ser 115 120 125Ser Val
Arg Gly Ala Val Asn Asp Leu Leu Ala Lys Trp His Gln Asp 130 135
140Tyr Gly Gln Val Asn Asn Val Pro Ala Arg Met Gln Tyr Glu Lys
Ile145 150 155 160Thr Ala His Ser Met Glu Gln Leu Lys Val Lys Phe
Gly Ser Asp Phe 165 170 175Glu Lys Thr Gly Asn Ser Leu Asp Ile Asp
Phe Asn Ser Val His Ser 180 185 190Gly Leu Lys Gln Ile Gln Ile Val
Asn Phe Lys Gln Ile Tyr Tyr Thr 195 200 205Val Ser Val Asp Ala Val
Arg Asn Pro Gly Asp Val Phe Gln Asp Thr 210 215 220Val Thr Val Glu
Asp Leu Lys Gln Arg Gly Ile Ser Ala Glu Arg Pro225 230 235 240Leu
Val Tyr Ile Ser Ser Val Ala Tyr Gly Arg Gln Val Tyr Leu Lys 245 250
255Leu Glu Thr Thr Ser Lys Ser Asp Glu Val Glu Ala Ala Phe Glu Ala
260 265 270Leu Ile Lys Gly Val Lys Val Ala Pro Gln Thr Glu Trp Lys
Gln Ile 275 280 285Leu Asp Asn Thr Glu Val Lys Ala Val Ile Leu Gly
Gly Asp Pro Ser 290 295 300Ser Gly Ala Arg Val Val Thr Gly Lys Val
Asp Met Val Asp Asp Leu305 310 315 320Ile Gln Glu Gly Ser Arg Phe
Thr Ala Asp His Pro Gly Leu Pro Ile 325 330 335Ser Tyr Thr Thr Ser
Phe Leu Arg Asp Asn Val Val Ala Thr Phe Gln 340 345 350Asn Ser Thr
Asp Tyr Val Glu Thr Lys Val Thr Ala Tyr Arg Asn Gly 355 360 365Asp
Leu Leu Leu Asp His Ser Gly Ala Tyr Val Ala Gln Tyr Tyr Ile 370 375
380Thr Trp Asp Glu Leu Ser Tyr Asp His Gln Gly Lys Glu Val Leu
Thr385 390 395 400Pro Lys Ala Trp Asp Arg Asn Gly Gln Asp Leu Thr
Ala His Phe Thr 405 410 415Thr Ser Ile Pro Leu Lys Gly Asn Val Arg
Asn Leu Ser Val Lys Ile 420 425 430Arg Glu Cys Thr Gly Leu Ala Trp
Glu Trp Trp Arg Thr Val Tyr Glu 435 440 445Lys Thr Asp Leu Pro Leu
Val Arg Lys Arg Thr Ile Ser Ile Trp Gly 450 455 460Thr Thr Leu Tyr
Pro Gln Val Glu Asp Lys Val Glu Asn465 470 47518477PRTArtificial
SequenceSynthetic polypeptide 18His His His His His His Gly Ser Ala
Asn Lys Ala Val Asn Asp Phe1 5 10 15Ile Leu Ala Met Asn Tyr Asp Lys
Lys Lys Leu Leu Thr His Gln Gly 20 25 30Glu Ser Ile Glu Asn Arg Phe
Ile Lys Glu Gly Asn Gln Leu Pro Asp 35 40 45Glu Phe Val Val Ile Glu
Arg Lys Lys Arg Ser Leu Ser Thr Ser Thr 50 55 60Ser Asp Ile Ser Val
Thr Ala Thr Asn Asp Ser Arg Leu Tyr Pro Gly65 70 75 80Ala Leu Leu
Val Val Asp Glu Thr Leu Leu Glu Asn Asn Pro Thr Leu 85 90 95Leu Ala
Val Asp Arg Ala Pro Met Thr Tyr Ser Ile Phe Leu Pro Gly 100 105
110Leu Gln Ser Ser Asp Ser Phe Leu Gln Val Glu Asp Pro Ser Asn Ser
115 120 125Ser Val Arg Gly Ala Val Asn Asp Leu Leu Ala Lys Trp His
Gln Asp 130 135 140Tyr Gly Gln Val Asn Asn Val Pro Ala Arg Met Gln
Tyr Glu Lys Ile145 150 155 160Thr Ala His Ser Met Glu Gln Leu Lys
Val Lys Phe Gly Ser Asp Phe 165 170 175Glu Lys Thr Gly Asn Ser Leu
Asp Ile Asp Phe Asn Ser Val His Ser 180 185 190Gly Glu Lys Gln Ile
Gln Ile Val Asn Phe Lys Gln Ile Tyr Tyr Thr 195 200 205Val Ser Val
Asp Ala Val Arg Asn Pro Gly Asp Val Phe Gln Asp Thr 210 215 220Val
Thr Val Glu Asp Leu Lys Gln Arg Gly Ile Ser Ala Glu Arg Pro225 230
235 240Leu Val Tyr Ile Ser Ser Val Ala Tyr Gly Arg Gln Val Tyr Leu
Lys 245 250 255Leu Glu Thr Thr Ser Lys Ser Asp Glu Val Glu Ala Ala
Phe Glu Ala 260 265 270Leu Ile Lys Gly Val Lys Val Ala Pro Gln Thr
Glu Trp Lys Gln Ile 275 280 285Leu Asp Asn Thr Glu Val Lys Ala Val
Ile Leu Gly Gly Asp Pro Ser 290 295 300Ser Gly Ala Arg Val Val Thr
Gly Lys Val Asp Met Val Asp Asp Leu305 310 315 320Ile Gln Glu Gly
Ser Arg Phe Thr Ala Asp His Pro Gly Leu Pro Ile 325 330 335Ser Tyr
Thr Thr Ser Phe Leu Arg Asp Asn Val Val Ala Thr Phe Gln 340 345
350Asn Ser Thr Asp Tyr Val Glu Thr Lys Val Thr Ala Tyr Arg Asn Gly
355 360 365Asp Leu Leu Leu Asp His Ser Gly Ala Tyr Val Ala Gln Tyr
Tyr Ile 370 375 380Thr Trp Asp Glu Leu Ser Tyr Asp His Gln Gly Lys
Glu Val Leu Thr385 390 395 400Pro Lys Ala Trp Asp Arg Asn Gly Gln
Asp Leu Thr Ala His Phe Thr 405 410 415Thr Ser Ile Pro Leu Lys Gly
Asn Val Arg Asn Leu Ser Val Lys Ile 420 425 430Arg Glu Cys Thr Gly
Leu Ala Trp Glu Trp Trp Arg Thr Val Tyr Glu 435 440 445Lys Thr Asp
Leu Pro Leu Val Arg Lys Arg Thr Ile Ser Ile Trp Gly 450 455 460Thr
Thr Leu Tyr Pro Gln Val Glu Asp Lys Val Glu Asn465 470
47519477PRTArtificial SequenceSynthetic polypeptide 19His His His
His His His Gly Ser Ala Asn Lys Ala Val Asn Asp Phe1 5 10 15Ile Leu
Ala Met Asn Tyr Asp Lys Lys Lys Leu Leu Thr His Gln Gly 20 25 30Glu
Ser Ile Glu Asn Arg Phe Ile Lys Glu Gly Asn Gln Leu Pro Asp 35 40
45Glu Phe Val Val Ile Glu Arg Lys Lys Arg Ser Leu Ser Thr Ser Thr
50 55 60Ser Asp Ile Ser Val Thr Ala Thr Asn Asp Ser Arg Leu Tyr Pro
Gly65 70 75 80Ala Leu Leu Val Val Asp Glu Thr Leu Leu Glu Asn Asn
Pro Thr Leu 85 90 95Leu Ala Val Asp Arg Ala Pro Met Thr Tyr Ser Ile
Phe Leu Pro Gly 100 105 110Leu Ala Ser Ser Asp Ser Phe Leu Gln Val
Glu Asp Pro Ser Phe Ser 115 120 125Ser Val Arg Gly Ala Val Asn Asp
Leu Leu Ala Lys Trp His Gln Asp 130 135 140Tyr Gly Gln Val Asn Asn
Val Pro Ala Arg Met Gln Tyr Glu Lys Ile145 150 155 160Thr Ala His
Ser Met Glu Gln Leu Lys Val Lys Phe Gly Ser Asp Phe 165 170 175Glu
Lys Thr Gly Asn Ser Leu Asp Ile Asp Phe Asn Ser Val His Ser 180 185
190Gly Glu Lys Gln Ile Gln Ile Val Asn Phe Lys Gln Ile Tyr Tyr Thr
195 200 205Val Ser Val Asp Ala Val Arg Asn Pro Gly Asp Val Phe Gln
Asp Thr 210 215 220Val Thr Val Glu Asp Leu Lys Gln Arg Gly Ile Ser
Ala Glu Arg Pro225 230 235 240Leu Val Tyr Ile Ser Ser Val Ala Tyr
Gly Arg Gln Val Tyr Leu Lys 245 250 255Leu Glu Thr Thr Ser Lys Ser
Asp Glu Val Glu Ala Ala Phe Glu Ala 260
265 270Leu Ile Lys Gly Val Lys Val Ala Pro Gln Thr Glu Trp Lys Gln
Ile 275 280 285Leu Asp Asn Thr Glu Val Lys Ala Val Ile Leu Gly Gly
Asp Pro Ser 290 295 300Ser Gly Ala Arg Val Val Thr Gly Lys Val Asp
Met Val Asp Asp Leu305 310 315 320Ile Gln Glu Gly Ser Arg Phe Thr
Ala Asp His Pro Gly Leu Pro Ile 325 330 335Ser Tyr Thr Thr Ser Phe
Leu Arg Asp Asn Val Val Ala Thr Phe Gln 340 345 350Asn Ser Thr Asp
Tyr Val Glu Thr Lys Val Thr Ala Tyr Arg Asn Gly 355 360 365Asp Leu
Leu Leu Asp His Ser Gly Ala Tyr Val Ala Gln Tyr Tyr Ile 370 375
380Thr Trp Asp Glu Leu Ser Tyr Asp His Gln Gly Lys Glu Val Leu
Thr385 390 395 400Pro Lys Ala Trp Asp Arg Asn Gly Gln Asp Leu Thr
Ala His Phe Thr 405 410 415Thr Ser Ile Pro Leu Lys Gly Asn Val Arg
Asn Leu Ser Val Lys Ile 420 425 430Arg Glu Cys Thr Gly Leu Ala Trp
Glu Trp Trp Arg Thr Val Tyr Glu 435 440 445Lys Thr Asp Leu Pro Leu
Val Arg Lys Arg Thr Ile Ser Ile Trp Gly 450 455 460Thr Thr Leu Tyr
Pro Gln Val Glu Asp Lys Val Glu Asn465 470 47520477PRTArtificial
SequenceSynthetic polypeptide 20His His His His His His Gly Ser Ala
Asn Lys Ala Val Asn Asp Phe1 5 10 15Ile Leu Ala Met Asn Tyr Asp Lys
Lys Lys Leu Leu Thr His Gln Gly 20 25 30Glu Ser Ile Glu Asn Arg Phe
Ile Lys Glu Gly Asn Gln Leu Pro Asp 35 40 45Glu Phe Val Val Ile Glu
Arg Lys Lys Arg Ser Leu Ser Thr Ser Thr 50 55 60Ser Asp Ile Ser Val
Thr Ala Thr Asn Asp Ser Arg Leu Tyr Pro Gly65 70 75 80Ala Leu Leu
Val Val Asp Glu Thr Leu Leu Glu Asn Asn Pro Thr Leu 85 90 95Leu Ala
Val Asp Arg Ala Pro Met Thr Tyr Ser Ile Asp Leu Pro Gly 100 105
110Leu Gln Ser Ser Asp Ser Phe Leu Gln Val Glu Asp Pro Ser Phe Ser
115 120 125Ser Val Arg Gly Ala Val Asn Asp Leu Leu Ala Lys Trp His
Gln Asp 130 135 140Tyr Gly Gln Val Asn Asn Val Pro Ala Arg Met Gln
Tyr Glu Lys Ile145 150 155 160Thr Ala His Ser Met Glu Gln Leu Lys
Val Lys Phe Gly Ser Asp Phe 165 170 175Glu Lys Thr Gly Asn Ser Leu
Asp Ile Asp Phe Asn Ser Val His Ser 180 185 190Gly Glu Lys Gln Ile
Gln Ile Val Asn Phe Lys Gln Ile Tyr Tyr Thr 195 200 205Val Ser Val
Asp Ala Val Arg Asn Pro Gly Asp Val Phe Gln Asp Thr 210 215 220Val
Thr Val Glu Asp Leu Lys Gln Arg Gly Ile Ser Ala Glu Arg Pro225 230
235 240Leu Val Tyr Ile Ser Ser Val Ala Tyr Gly Arg Gln Val Tyr Leu
Lys 245 250 255Leu Glu Thr Thr Ser Lys Ser Asp Glu Val Glu Ala Ala
Phe Glu Ala 260 265 270Leu Ile Lys Gly Val Lys Val Ala Pro Gln Thr
Glu Trp Lys Gln Ile 275 280 285Leu Asp Asn Thr Glu Val Lys Ala Val
Ile Leu Gly Gly Asp Pro Ser 290 295 300Ser Gly Ala Arg Val Val Thr
Gly Lys Val Asp Met Val Asp Asp Leu305 310 315 320Ile Gln Glu Gly
Ser Arg Phe Thr Ala Asp His Pro Gly Leu Pro Ile 325 330 335Ser Tyr
Thr Thr Ser Phe Leu Arg Asp Asn Val Val Ala Thr Phe Gln 340 345
350Asn Ser Thr Asp Tyr Val Glu Thr Lys Val Thr Ala Tyr Arg Asn Gly
355 360 365Asp Leu Leu Leu Asp His Ser Gly Ala Tyr Val Ala Gln Tyr
Tyr Ile 370 375 380Thr Trp Asp Glu Leu Ser Tyr Asp His Gln Gly Lys
Glu Val Leu Thr385 390 395 400Pro Lys Ala Trp Asp Arg Asn Gly Gln
Asp Leu Thr Ala His Phe Thr 405 410 415Thr Ser Ile Pro Leu Lys Gly
Asn Val Arg Asn Leu Ser Val Lys Ile 420 425 430Arg Glu Cys Thr Gly
Leu Ala Trp Glu Trp Trp Arg Thr Val Tyr Glu 435 440 445Lys Thr Asp
Leu Pro Leu Val Arg Lys Arg Thr Ile Ser Ile Trp Gly 450 455 460Thr
Thr Leu Tyr Pro Gln Val Glu Asp Lys Val Glu Asn465 470
47521477PRTArtificial SequenceSynthetic polypeptide 21His His His
His His His Gly Ser Ala Asn Lys Ala Val Asn Asp Phe1 5 10 15Ile Leu
Ala Met Asn Tyr Asp Lys Lys Lys Leu Leu Thr His Gln Gly 20 25 30Glu
Ser Ile Glu Asn Arg Phe Ile Lys Glu Gly Asn Gln Leu Pro Asp 35 40
45Glu Phe Val Val Ile Glu Arg Lys Lys Arg Ser Leu Ser Thr Ser Thr
50 55 60Ser Asp Ile Ser Val Thr Ala Thr Asn Asp Ser Arg Leu Tyr Pro
Gly65 70 75 80Ala Leu Leu Val Val Asp Glu Thr Leu Leu Glu Asn Asn
Pro Thr Leu 85 90 95Leu Ala Val Asp Arg Ala Pro Met Thr Tyr Ser Ile
Phe Leu Pro Gly 100 105 110Leu Gln Ser Ser Asp Ser Phe Leu Gln Val
Glu Asp Pro Ser Phe Ser 115 120 125Ser Val Arg Gly Ala Val Asn Asp
Leu Leu Ala Lys Trp His Gln Asp 130 135 140Tyr Gly Gln Val Asn Asn
Val Pro Ala Arg Met Gln Tyr Glu Lys Ile145 150 155 160Thr Ala His
Ser Met Glu Gln Leu Lys Val Lys Phe Gly Ser Asp Phe 165 170 175Glu
Lys Thr Gly Asn Ser Leu Asp Ile Asp Phe Asn Ser Val His Ser 180 185
190Gly Glu Lys Gln Ile Gln Ile Val Asn Phe Lys Gln Ile Tyr Tyr Thr
195 200 205Val Ser Val Asp Ala Val Arg Asn Pro Gly Asp Val Phe Gln
Asp Thr 210 215 220Val Thr Val Glu Asp Leu Lys Gln Arg Gly Ile Ser
Ala Glu Arg Pro225 230 235 240Leu Val Tyr Ile Ser Ser Val Ala Tyr
Gly Arg Gln Val Tyr Leu Lys 245 250 255Leu Glu Thr Thr Ser Lys Ser
Asp Glu Val Glu Ala Ala Phe Glu Ala 260 265 270Leu Ile Lys Gly Val
Lys Val Ala Pro Gln Thr Glu Trp Lys Gln Ile 275 280 285Leu Asp Asn
Thr Glu Val Lys Ala Val Ile Leu Gly Gly Asp Pro Ser 290 295 300Ser
Gly Ala Arg Val Val Thr Gly Lys Val Asp Met Val Asp Asp Leu305 310
315 320Ile Gln Glu Gly Ser Arg Phe Thr Ala Asp His Pro Gly Leu Pro
Ile 325 330 335Ser Tyr Thr Thr Ser Phe Leu Arg Asp Asn Val Val Ala
Thr Phe Gln 340 345 350Asn Ser Thr Asp Tyr Val Glu Thr Lys Val Thr
Ala Tyr Arg Asn Gly 355 360 365Asp Leu Leu Leu Asp His Ser Gly Ala
Tyr Val Ala Gln Tyr Tyr Ile 370 375 380Thr Trp Asp Glu Leu Ser Tyr
Asp His Gln Gly Lys Glu Val Leu Thr385 390 395 400Pro Lys Ala Trp
Asp Arg Asn Gly Gln Asp Leu Thr Ala His Phe Thr 405 410 415Thr Ser
Ile Pro Leu Lys Gly Asn Val Arg Asn Leu Ser Val Lys Ile 420 425
430Arg Glu Cys Thr Gly Leu Ala Trp Glu Trp Trp Arg Thr Val Tyr Glu
435 440 445Lys Thr Asp Leu Pro Leu Val Arg Lys Arg Thr Ile Ser Ile
Trp Gly 450 455 460Thr Thr Leu Tyr Pro Gln Val Glu Asp Lys Val Glu
Asn465 470 47522477PRTArtificial SequenceSynthetic polypeptide
22His His His His His His Gly Ser Ala Asn Lys Ala Val Asn Asp Phe1
5 10 15Ile Leu Ala Met Asn Tyr Asp Lys Lys Lys Leu Leu Thr His Gln
Gly 20 25 30Glu Ser Ile Glu Asn Arg Phe Ile Lys Glu Gly Asn Gln Leu
Pro Asp 35 40 45Glu Phe Val Val Ile Glu Arg Lys Lys Arg Ser Leu Ser
Thr Ser Thr 50 55 60Ser Asp Ile Ser Val Thr Ala Thr Asn Asp Ser Arg
Leu Tyr Pro Gly65 70 75 80Ala Leu Leu Val Val Asp Glu Thr Leu Leu
Glu Asn Asn Pro Thr Leu 85 90 95Leu Ala Val Asp Arg Ala Pro Met Thr
Tyr Ser Ile Phe Leu Pro Gly 100 105 110Leu Gln Ser Ser Asp Ser Phe
Leu Gln Val Glu Asp Pro Ser Phe Ser 115 120 125Ser Val Arg Gly Ala
Val Asn Asp Leu Leu Ala Lys Trp His Gln Asp 130 135 140Tyr Gly Gln
Val Asn Asn Val Pro Ala Arg Met Gln Tyr Glu Lys Ile145 150 155
160Thr Ala His Ser Met Glu Gln Leu Lys Val Lys Phe Gly Ser Asp Phe
165 170 175Glu Lys Thr Gly Asn Ser Leu Asp Ile Asp Phe Asn Ser Val
His Ser 180 185 190Gly Leu Lys Gln Ile Gln Ile Val Asn Phe Lys Gln
Ile Tyr Tyr Thr 195 200 205Val Ser Val Asp Ala Val Arg Asn Pro Gly
Asp Val Phe Gln Asp Thr 210 215 220Val Thr Val Glu Asp Leu Lys Gln
Arg Gly Ile Ser Ala Glu Arg Pro225 230 235 240Leu Val Tyr Ile Ser
Ser Val Ala Tyr Gly Arg Gln Val Tyr Leu Lys 245 250 255Leu Glu Thr
Thr Ser Lys Ser Asp Glu Val Glu Ala Ala Phe Glu Ala 260 265 270Leu
Ile Lys Gly Val Lys Val Ala Pro Gln Thr Glu Trp Lys Gln Ile 275 280
285Leu Asp Asn Thr Glu Val Lys Ala Val Ile Leu Gly Gly Asp Pro Ser
290 295 300Ser Gly Ala Arg Val Val Thr Gly Lys Val Asp Met Val Asp
Asp Leu305 310 315 320Ile Gln Glu Gly Ser Arg Phe Thr Ala Asp His
Pro Gly Leu Pro Ile 325 330 335Ser Tyr Thr Thr Ser Phe Leu Arg Asp
Asn Val Val Ala Thr Phe Gln 340 345 350Asn Ser Thr Asp Tyr Val Glu
Thr Lys Val Thr Ala Tyr Arg Asn Gly 355 360 365Asp Leu Leu Leu Asp
His Ser Gly Ala Tyr Val Ala Gln Tyr Tyr Ile 370 375 380Thr Trp Asp
Glu Leu Ser Tyr Asp His Gln Gly Lys Glu Val Leu Thr385 390 395
400Pro Lys Ala Trp Asp Arg Asn Gly Gln Asp Leu Thr Ala His Phe Thr
405 410 415Thr Ser Ile Pro Leu Lys Gly Asn Val Arg Asn Leu Ser Val
Lys Ile 420 425 430Arg Glu Cys Thr Gly Leu Ala Trp Glu Trp Trp Arg
Thr Val Tyr Glu 435 440 445Lys Thr Asp Leu Pro Leu Val Arg Lys Arg
Thr Ile Ser Ile Trp Gly 450 455 460Thr Thr Leu Tyr Pro Gln Val Glu
Asp Lys Val Glu Asn465 470 47523477PRTArtificial SequenceSynthetic
polypeptide 23His His His His His His Gly Ser Ala Asn Lys Ala Val
Asn Asp Phe1 5 10 15Ile Leu Ala Met Asn Tyr Asp Lys Lys Lys Leu Leu
Thr His Gln Gly 20 25 30Glu Ser Ile Glu Asn Arg Phe Ile Lys Glu Gly
Asn Gln Leu Pro Asp 35 40 45Glu Phe Val Val Ile Glu Arg Lys Lys Arg
Ser Leu Ser Thr Ser Thr 50 55 60Ser Asp Ile Ser Val Thr Ala Thr Asn
Asp Ser Arg Leu Tyr Pro Gly65 70 75 80Ala Leu Leu Val Val Asp Glu
Thr Leu Leu Glu Asn Asn Pro Thr Leu 85 90 95Leu Ala Val Asp Arg Ala
Pro Met Thr Tyr Ser Ile Asp Leu Pro Gly 100 105 110Leu Ala Ser Ser
Asp Ser Phe Leu Gln Val Glu Asp Pro Ser Asn Ser 115 120 125Ser Val
Arg Gly Ala Val Asn Asp Leu Leu Ala Lys Trp His Gln Asp 130 135
140Tyr Gly Gln Val Asn Asn Val Pro Ala Arg Met Gln Tyr Glu Lys
Ile145 150 155 160Thr Ala His Ser Met Glu Gln Leu Lys Val Lys Phe
Gly Ser Asp Phe 165 170 175Glu Lys Thr Gly Asn Ser Leu Asp Ile Asp
Phe Asn Ser Val His Ser 180 185 190Gly Glu Lys Gln Ile Gln Ile Val
Asn Phe Lys Gln Ile Tyr Tyr Thr 195 200 205Val Ser Val Phe Ala Val
Arg Asn Pro Gly Asp Val Phe Gln Asp Thr 210 215 220Val Thr Val Glu
Asp Leu Lys Gln Arg Gly Ile Ser Ala Glu Arg Pro225 230 235 240Leu
Val Tyr Ile Ser Ser Val Ala Tyr Gly Arg Gln Val Tyr Leu Lys 245 250
255Leu Glu Thr Thr Ser Lys Ser Asp Glu Val Glu Ala Ala Phe Glu Ala
260 265 270Leu Ile Lys Gly Val Lys Val Ala Pro Gln Thr Glu Trp Lys
Gln Ile 275 280 285Leu Asp Asn Thr Glu Val Lys Ala Val Ile Leu Gly
Gly Asp Pro Ser 290 295 300Ser Gly Ala Arg Val Val Thr Gly Lys Val
Asp Met Val Asp Asp Leu305 310 315 320Ile Gln Glu Gly Ser Arg Phe
Thr Ala Asp His Pro Gly Leu Pro Ile 325 330 335Ser Tyr Thr Thr Ser
Phe Leu Arg Asp Asn Val Val Ala Thr Phe Gln 340 345 350Asn Ser Thr
Asp Tyr Val Glu Thr Lys Val Thr Ala Tyr Arg Asn Gly 355 360 365Asp
Leu Leu Leu Asp His Ser Gly Ala Tyr Val Ala Gln Tyr Tyr Ile 370 375
380Thr Trp Asp Glu Leu Ser Tyr Asp His Gln Gly Lys Glu Val Leu
Thr385 390 395 400Pro Lys Ala Trp Asp Arg Asn Gly Gln Asp Leu Thr
Ala His Phe Thr 405 410 415Thr Ser Ile Pro Leu Lys Gly Asn Val Arg
Asn Leu Ser Val Lys Ile 420 425 430Arg Glu Cys Thr Gly Leu Ala Trp
Glu Trp Trp Arg Thr Val Tyr Glu 435 440 445Lys Thr Asp Leu Pro Leu
Val Arg Lys Arg Thr Ile Ser Ile Trp Gly 450 455 460Thr Thr Leu Tyr
Pro Gln Val Glu Asp Lys Val Glu Asn465 470 47524477PRTArtificial
SequenceSynthetic polypeptide 24His His His His His His Gly Ser Ala
Asn Lys Ala Val Asn Asp Phe1 5 10 15Ile Leu Ala Met Asn Tyr Asp Lys
Lys Lys Leu Leu Thr His Gln Gly 20 25 30Glu Ser Ile Glu Asn Arg Phe
Ile Lys Glu Gly Asn Gln Leu Pro Asp 35 40 45Glu Phe Val Val Ile Glu
Arg Lys Lys Arg Ser Leu Ser Thr Ser Thr 50 55 60Ser Asp Ile Ser Val
Thr Ala Thr Asn Asp Ser Arg Leu Tyr Pro Gly65 70 75 80Ala Leu Leu
Val Val Asp Glu Thr Leu Leu Glu Asn Asn Pro Thr Leu 85 90 95Leu Ala
Val Asp Arg Ala Pro Met Thr Tyr Ser Ile Asp Leu Pro Gly 100 105
110Leu Ala Ser Ser Asp Ser Phe Leu Gln Val Glu Asp Pro Ser Asn Ser
115 120 125Ser Val Arg Gly Ala Val Asn Asp Leu Leu Ala Lys Trp His
Gln Asp 130 135 140Tyr Gly Gln Val Asn Asn Val Pro Ala Arg Met Gln
Tyr Glu Lys Ile145 150 155 160Thr Ala His Ser Met Glu Gln Leu Lys
Val Lys Phe Gly Ser Asp Phe 165 170 175Glu Lys Thr Gly Asn Ser Leu
Asp Ile Asp Phe Asn Ser Val His Ser 180 185 190Gly Glu Lys Gln Ile
Gln Ile Val Asn Phe Lys Gln Ile Tyr Tyr Thr 195 200 205Val Ser Val
Pro Ala Val Arg Asn Pro Gly Asp Val Phe Gln Asp Thr 210 215 220Val
Thr Val Glu Asp Leu Lys Gln Arg Gly Ile Ser Ala Glu Arg Pro225 230
235 240Leu Val Tyr Ile Ser Ser Val Ala Tyr Gly Arg Gln Val Tyr Leu
Lys 245 250 255Leu Glu Thr Thr Ser Lys Ser Asp Glu Val Glu Ala Ala
Phe Glu Ala 260 265 270Leu Ile Lys Gly Val Lys Val Ala Pro Gln Thr
Glu Trp Lys Gln Ile 275 280 285Leu Asp Asn Thr Glu Val Lys Ala Val
Ile Leu Gly Gly Asp Pro Ser 290 295 300Ser Gly Ala Arg Val Val Thr
Gly Lys Val Asp Met Val Asp Asp Leu305 310 315 320Ile Gln Glu Gly
Ser Arg Phe Thr Ala Asp His Pro Gly Leu Pro Ile
325 330 335Ser Tyr Thr Thr Ser Phe Leu Arg Asp Asn Val Val Ala Thr
Phe Gln 340 345 350Asn Ser Thr Asp Tyr Val Glu Thr Lys Val Thr Ala
Tyr Arg Asn Gly 355 360 365Asp Leu Leu Leu Asp His Ser Gly Ala Tyr
Val Ala Gln Tyr Tyr Ile 370 375 380Thr Trp Asp Glu Leu Ser Tyr Asp
His Gln Gly Lys Glu Val Leu Thr385 390 395 400Pro Lys Ala Trp Asp
Arg Asn Gly Gln Asp Leu Thr Ala His Phe Thr 405 410 415Thr Ser Ile
Pro Leu Lys Gly Asn Val Arg Asn Leu Ser Val Lys Ile 420 425 430Arg
Glu Cys Thr Gly Leu Ala Trp Glu Trp Trp Arg Thr Val Tyr Glu 435 440
445Lys Thr Asp Leu Pro Leu Val Arg Lys Arg Thr Ile Ser Ile Trp Gly
450 455 460Thr Thr Leu Tyr Pro Gln Val Glu Asp Lys Val Glu Asn465
470 47525477PRTArtificial SequenceSynthetic polypeptide 25His His
His His His His Gly Ser Ala Asn Lys Ala Val Asn Asp Phe1 5 10 15Ile
Leu Ala Met Asn Tyr Asp Lys Lys Lys Leu Leu Thr His Gln Gly 20 25
30Glu Ser Ile Glu Asn Arg Phe Ile Lys Glu Gly Asn Gln Leu Pro Asp
35 40 45Glu Phe Val Val Ile Glu Arg Lys Lys Arg Ser Leu Ser Thr Ser
Thr 50 55 60Ser Asp Ile Ser Val Thr Ala Thr Asn Asp Ser Arg Leu Tyr
Pro Gly65 70 75 80Ala Leu Leu Val Val Asp Glu Thr Leu Leu Glu Asn
Asn Pro Thr Leu 85 90 95Leu Ala Val Asp Arg Ala Pro Met Thr Tyr Ser
Ile Asp Leu Pro Gly 100 105 110Leu Ala Ser Ser Asp Ser Phe Leu Gln
Val Glu Asp Pro Ser Asn Ser 115 120 125Ser Val Arg Gly Ala Val Asn
Asp Leu Leu Ala Lys Trp His Gln Asp 130 135 140Tyr Gly Gln Val Asn
Asn Val Pro Ala Arg Met Gln Tyr Glu Lys Ile145 150 155 160Thr Ala
His Ser Met Glu Gln Leu Lys Val Lys Phe Gly Ser Asp Phe 165 170
175Glu Lys Thr Gly Asn Ser Leu Asp Ile Asp Phe Asn Ser Val His Ser
180 185 190Gly Glu Lys Gln Ile Gln Ile Val Asn Phe Lys Gln Ile Tyr
Tyr Thr 195 200 205Val Ser Val Asp Ala Val Arg Asn Pro Gly Asp Val
Phe Gln Asp Thr 210 215 220Val Thr Val Glu Asp Leu Lys Gln Arg Gly
Ile Ser Ala Glu Arg Pro225 230 235 240Leu Val Tyr Ile Ser Ser Val
Ala Tyr Gly Arg Gln Val Tyr Leu Lys 245 250 255Leu Glu Thr Thr Ser
Lys Ser Asp Glu Val Glu Ala Ala Phe Glu Ala 260 265 270Leu Ile Lys
Gly Val Lys Val Ala Pro Gln Thr Glu Trp Lys Gln Ile 275 280 285Leu
Asp Asn Thr Glu Val Lys Ala Val Ile Leu Ile Gly Asp Pro Ser 290 295
300Ser Gly Ala Arg Val Val Thr Gly Lys Val Asp Met Val Asp Asp
Leu305 310 315 320Ile Gln Glu Gly Ser Arg Phe Thr Ala Asp His Pro
Gly Leu Pro Ile 325 330 335Ser Tyr Thr Thr Ser Phe Leu Arg Asp Asn
Val Val Ala Thr Phe Gln 340 345 350Asn Ser Thr Asp Tyr Val Glu Thr
Lys Val Thr Ala Tyr Arg Asn Gly 355 360 365Asp Leu Leu Leu Asp His
Ser Gly Ala Tyr Val Ala Gln Tyr Tyr Ile 370 375 380Thr Trp Asp Glu
Leu Ser Tyr Asp His Gln Gly Lys Glu Val Leu Thr385 390 395 400Pro
Lys Ala Trp Asp Arg Asn Gly Gln Asp Leu Thr Ala His Phe Thr 405 410
415Thr Ser Ile Pro Leu Lys Gly Asn Val Arg Asn Leu Ser Val Lys Ile
420 425 430Arg Glu Cys Thr Gly Leu Ala Trp Glu Trp Trp Arg Thr Val
Tyr Glu 435 440 445Lys Thr Asp Leu Pro Leu Val Arg Lys Arg Thr Ile
Ser Ile Trp Gly 450 455 460Thr Thr Leu Tyr Pro Gln Val Glu Asp Lys
Val Glu Asn465 470 47526477PRTArtificial SequenceSynthetic
polypeptide 26His His His His His His Gly Ser Ala Asn Lys Ala Val
Asn Asp Phe1 5 10 15Ile Leu Ala Met Asn Tyr Asp Lys Lys Lys Leu Leu
Thr His Gln Gly 20 25 30Glu Ser Ile Glu Asn Arg Phe Ile Lys Glu Gly
Asn Gln Leu Pro Asp 35 40 45Glu Phe Val Val Ile Glu Arg Lys Lys Arg
Ser Leu Ser Thr Ser Thr 50 55 60Ser Asp Ile Ser Val Thr Ala Thr Asn
Asp Ser Arg Leu Tyr Pro Gly65 70 75 80Ala Leu Leu Val Val Asp Glu
Thr Leu Leu Glu Asn Asn Pro Thr Leu 85 90 95Leu Ala Val Asp Arg Ala
Pro Met Thr Tyr Ser Ile Asp Leu Pro Gly 100 105 110Leu Ala Ser Ser
Asp Ser Phe Leu Gln Val Glu Asp Pro Ser Phe Ser 115 120 125Ser Val
Arg Gly Ala Val Asn Asp Leu Leu Ala Lys Trp His Gln Asp 130 135
140Tyr Gly Gln Val Asn Asn Val Pro Ala Arg Met Gln Tyr Glu Lys
Ile145 150 155 160Thr Ala His Ser Met Glu Gln Leu Lys Val Lys Phe
Gly Ser Asp Phe 165 170 175Glu Lys Thr Gly Asn Ser Leu Asp Ile Asp
Phe Asn Ser Val His Ser 180 185 190Gly Glu Lys Gln Ile Gln Ile Val
Asn Phe Lys Gln Ile Tyr Tyr Thr 195 200 205Val Ser Val Pro Ala Val
Arg Asn Pro Gly Asp Val Phe Gln Asp Thr 210 215 220Val Thr Val Glu
Asp Leu Lys Gln Arg Gly Ile Ser Ala Glu Arg Pro225 230 235 240Leu
Val Tyr Ile Ser Ser Val Ala Tyr Gly Arg Gln Val Tyr Leu Lys 245 250
255Leu Glu Thr Thr Ser Lys Ser Asp Glu Val Glu Ala Ala Phe Glu Ala
260 265 270Leu Ile Lys Gly Val Lys Val Ala Pro Gln Thr Glu Trp Lys
Gln Ile 275 280 285Leu Asp Asn Thr Glu Val Lys Ala Val Ile Leu Gly
Gly Asp Pro Ser 290 295 300Ser Gly Ala Arg Val Val Thr Gly Lys Val
Asp Met Val Asp Asp Leu305 310 315 320Ile Gln Glu Gly Ser Arg Phe
Thr Ala Asp His Pro Gly Leu Pro Ile 325 330 335Ser Tyr Thr Thr Ser
Phe Leu Arg Asp Asn Val Val Ala Thr Phe Gln 340 345 350Asn Ser Thr
Asp Tyr Val Glu Thr Lys Val Thr Ala Tyr Arg Asn Gly 355 360 365Asp
Leu Leu Leu Asp His Ser Gly Ala Tyr Val Ala Gln Tyr Tyr Ile 370 375
380Thr Trp Asp Glu Leu Ser Tyr Asp His Gln Gly Lys Glu Val Leu
Thr385 390 395 400Pro Lys Ala Trp Asp Arg Asn Gly Gln Asp Leu Thr
Ala His Phe Thr 405 410 415Thr Ser Ile Pro Leu Lys Gly Asn Val Arg
Asn Leu Ser Val Lys Ile 420 425 430Arg Glu Cys Thr Gly Leu Ala Trp
Glu Trp Trp Arg Thr Val Tyr Glu 435 440 445Lys Thr Asp Leu Pro Leu
Val Arg Lys Arg Thr Ile Ser Ile Trp Gly 450 455 460Thr Thr Leu Tyr
Pro Gln Val Glu Asp Lys Val Glu Asn465 470 47527491PRTArtificial
SequenceSynthetic polypeptide 27Met Glu Thr Pro Ala Gln Leu Leu Phe
Leu Leu Leu Leu Trp Leu Pro1 5 10 15Asp Thr Thr Gly Met Ala Asn Lys
Ala Val Asn Asp Phe Ile Leu Ala 20 25 30Met Asn Tyr Asp Lys Lys Lys
Leu Leu Thr His Gln Gly Glu Ser Ile 35 40 45Glu Asn Arg Phe Ile Lys
Glu Gly Asn Gln Leu Pro Asp Glu Phe Val 50 55 60Val Ile Glu Arg Lys
Lys Arg Ser Leu Ser Thr Asp Thr Ser Asp Ile65 70 75 80Ser Val Thr
Ala Thr Asn Asp Ala Arg Leu Tyr Pro Gly Ala Leu Leu 85 90 95Val Val
Asp Glu Thr Leu Leu Glu Asn Asn Pro Ile Leu Leu Ala Val 100 105
110Asp Arg Ala Pro Met Thr Tyr Ser Ile Asp Leu Pro Gly Leu Ala Ser
115 120 125Ser Asp Ser Phe Leu Gln Val Glu Asp Pro Ser Asn Ser Ala
Val Arg 130 135 140Gly Ala Val Asn Asp Leu Leu Ala Lys Trp His Gln
Asp Tyr Gly Gln145 150 155 160Val Asn Asn Val Pro Ala Arg Met Gln
Tyr Glu Lys Ile Thr Ala His 165 170 175Ser Met Glu Gln Leu Lys Val
Lys Phe Gly Ser Asp Phe Glu Lys Thr 180 185 190Gly Asn Ser Leu Asp
Ile Asp Phe Asn Ser Val His Ser Gly Glu Lys 195 200 205Gln Ile Gln
Ile Val Asn Phe Lys Gln Ile Tyr Tyr Thr Val Ser Val 210 215 220Arg
Ala Val Lys Asn Pro Gly Asp Val Phe Gln Asp Thr Val Thr Val225 230
235 240Glu Asp Leu Lys Gln Arg Gly Ile Ser Ala Glu Arg Pro Leu Val
Tyr 245 250 255Ile Ser Ser Val Ala Tyr Gly Arg Gln Val Tyr Leu Lys
Leu Glu Thr 260 265 270Thr Ser Lys Ser Asp Glu Val Glu Ala Ala Phe
Glu Ala Leu Ile Lys 275 280 285Gly Val Lys Val Ala Pro Gln Thr Glu
Trp Lys Gln Ile Leu Asp Asn 290 295 300Thr Glu Val Lys Ala Val Ile
Leu Gly Gly Asp Pro Ser Ser Gly Ala305 310 315 320Arg Val Val Thr
Gly Lys Val Asp Met Val Glu Asp Leu Ile Gln Glu 325 330 335Gly Ser
Arg Phe Thr Ala Asp His Pro Gly Leu Pro Ile Ser Tyr Thr 340 345
350Thr Ser Phe Leu Arg Asp Asn Val Val Ala Thr Phe Gln Asn Ser Ala
355 360 365Asp Tyr Val Glu Thr Lys Val Thr Ala Tyr Arg Asn Gly Asp
Leu Leu 370 375 380Leu Asp His Ser Gly Ala Tyr Val Ala Gln Tyr Tyr
Ile Thr Trp Asp385 390 395 400Glu Leu Ser Tyr Asp His Gln Gly Lys
Glu Val Leu Thr Pro Lys Ala 405 410 415Trp Asp Arg Asn Gly Gln Asp
Leu Thr Ala His Phe Thr Thr Ser Ile 420 425 430Pro Leu Lys Gly Asn
Val Arg Asn Leu Ser Val Lys Ile Arg Glu Cys 435 440 445Thr Gly Leu
Ala Trp Glu Trp Trp Arg Thr Val Tyr Glu Lys Thr Asp 450 455 460Leu
Pro Leu Val Arg Lys Arg Thr Ile Ser Ile Trp Gly Thr Thr Leu465 470
475 480Tyr Pro Gln Val Glu Asp Lys Val Glu Asn Asp 485
49028491PRTArtificial SequenceSynthetic polypeptide 28Met Glu Thr
Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro1 5 10 15Asp Thr
Thr Gly Met Ala Asn Lys Ala Val Asn Asp Phe Ile Leu Ala 20 25 30Met
Asn Tyr Asp Lys Lys Lys Leu Leu Thr His Gln Gly Glu Ser Ile 35 40
45Glu Asn Arg Phe Ile Lys Glu Gly Asn Gln Leu Pro Asp Glu Phe Val
50 55 60Val Ile Glu Arg Lys Lys Arg Ser Leu Ser Thr Asp Thr Ser Asp
Ile65 70 75 80Ser Val Thr Ala Thr Asn Asp Ala Arg Leu Tyr Pro Gly
Ala Leu Leu 85 90 95Val Val Asp Glu Thr Leu Leu Glu Asn Asn Pro Ile
Leu Leu Ala Val 100 105 110Asp Arg Ala Pro Met Thr Tyr Ser Ile Asp
Leu Pro Gly Leu Ala Ser 115 120 125Ser Asp Ser Phe Leu Gln Val Glu
Asp Pro Ser Asn Ser Ala Val Arg 130 135 140Gly Ala Val Asn Asp Leu
Leu Ala Lys Trp His Gln Asp Tyr Gly Gln145 150 155 160Val Asn Asn
Val Pro Ala Arg Met Gln Tyr Glu Lys Ile Thr Ala His 165 170 175Ser
Met Glu Gln Leu Lys Val Lys Phe Gly Ser Asp Phe Glu Lys Thr 180 185
190Gly Asn Ser Leu Asp Ile Asp Phe Asn Ser Val His Ser Gly Glu Lys
195 200 205Gln Ile Gln Ile Val Asn Phe Lys Gln Ile Tyr Tyr Thr Val
Ser Val 210 215 220Asp Ala Val Lys Asn Pro Gly Asp Val Phe Gln Asp
Thr Val Thr Val225 230 235 240Glu Asp Leu Lys Gln Arg Gly Ile Ser
Ala Glu Arg Pro Leu Val Tyr 245 250 255Ile Ser Ser Val Ala Tyr Gly
Arg Gln Val Tyr Leu Lys Leu Glu Thr 260 265 270Thr Ser Lys Ser Asp
Glu Val Glu Ala Ala Phe Glu Ala Leu Ile Lys 275 280 285Gly Val Lys
Val Ala Pro Gln Thr Glu Trp Lys Gln Ile Leu Asp Asn 290 295 300Thr
Glu Val Lys Ala Val Ile Leu Gly Gly Asp Pro Ser Ser Gly Ala305 310
315 320Arg Val Val Thr Gly Lys Val Asp Met Val Glu Asp Leu Ile Gln
Glu 325 330 335Gly Ser Arg Phe Thr Ala Asp His Pro Gly Leu Pro Ile
Ser Tyr Thr 340 345 350Thr Ser Phe Leu Arg Asp Asn Val Val Ala Thr
Phe Gln Asn Ser Ala 355 360 365Asp Tyr Val Glu Thr Lys Val Thr Ala
Tyr Arg Asn Gly Asp Leu Leu 370 375 380Leu Asp His Ser Gly Ala Tyr
Val Ala Gln Tyr Tyr Ile Thr Trp Asp385 390 395 400Glu Leu Ser Tyr
Asp His Gln Gly Lys Glu Val Leu Thr Pro Lys Ala 405 410 415Trp Asp
Arg Asn Gly Gln Asp Leu Thr Ala His Phe Thr Thr Ser Ile 420 425
430Pro Leu Lys Gly Asn Val Arg Asn Leu Ser Val Lys Ile Arg Glu Cys
435 440 445Thr Gly Leu Ala Trp Glu Trp Trp Arg Thr Val Tyr Glu Lys
Thr Asp 450 455 460Leu Pro Leu Val Arg Lys Arg Thr Ile Ser Ile Trp
Gly Thr Thr Asp465 470 475 480Tyr Pro Gln Val Glu Asp Lys Val Glu
Asn Asp 485 49029491PRTArtificial SequenceSynthetic polypeptide
29Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro1
5 10 15Asp Thr Thr Gly Met Ala Asn Lys Ala Val Asn Asp Phe Ile Leu
Ala 20 25 30Met Asn Tyr Asp Lys Lys Lys Leu Leu Thr His Gln Gly Glu
Ser Ile 35 40 45Glu Asn Arg Phe Ile Lys Glu Gly Asn Gln Leu Pro Asp
Glu Phe Val 50 55 60Val Ile Glu Arg Lys Lys Arg Ser Leu Ser Thr Asp
Thr Ser Asp Ile65 70 75 80Ser Val Thr Ala Cys Asn Asp Ala Arg Leu
Tyr Pro Gly Ala Leu Leu 85 90 95Val Val Asp Glu Thr Leu Leu Glu Asn
Asn Pro Ile Leu Leu Ala Val 100 105 110Asp Arg Ala Pro Met Thr Tyr
Ser Ile Asp Leu Pro Gly Leu Ala Ser 115 120 125Ser Asp Ser Phe Leu
Gln Val Glu Asp Pro Ser Asn Ser Ala Val Arg 130 135 140Gly Ala Val
Asn Asp Leu Leu Ala Lys Trp His Gln Asp Tyr Gly Gln145 150 155
160Val Asn Asn Val Pro Ala Arg Met Gln Tyr Glu Lys Ile Thr Ala His
165 170 175Ser Met Glu Gln Leu Lys Val Lys Phe Gly Ser Asp Phe Glu
Lys Thr 180 185 190Gly Asn Ser Leu Asp Ile Asp Phe Asn Ser Val His
Ser Gly Glu Lys 195 200 205Gln Ile Gln Ile Val Asn Phe Lys Gln Ile
Tyr Tyr Thr Val Ser Val 210 215 220Asp Ala Val Lys Asn Pro Gly Asp
Val Phe Gln Asp Thr Val Thr Val225 230 235 240Glu Asp Leu Lys Gln
Arg Gly Ile Ser Ala Glu Arg Pro Leu Val Tyr 245 250 255Ile Ser Ser
Val Ala Tyr Gly Arg Gln Val Tyr Leu Lys Leu Glu Thr 260 265 270Thr
Ser Lys Ser Asp Glu Val Glu Ala Ala Phe Glu Ala Leu Ile Lys 275 280
285Gly Val Lys Val Ala Pro Gln Thr Glu Trp Lys Gln Ile Leu Asp Asn
290 295 300Thr Glu Val Lys Ala Val Ile Leu Cys Gly Asp Pro Ser Ser
Gly Ala305 310 315 320Arg Val Val Thr Gly Lys Val Asp Met Val Glu
Asp Leu Ile Gln Glu 325 330 335Gly Ser Arg Phe Thr Ala Asp His Pro
Gly Leu Pro Ile Ser Tyr Thr 340 345 350Thr Ser Phe Leu Arg Asp Asn
Val Val Ala Thr
Phe Gln Asn Ser Ala 355 360 365Asp Tyr Val Glu Thr Lys Val Thr Ala
Tyr Arg Asn Gly Asp Leu Leu 370 375 380Leu Asp His Ser Gly Ala Tyr
Val Ala Gln Tyr Tyr Ile Thr Trp Asp385 390 395 400Glu Leu Ser Tyr
Asp His Gln Gly Lys Glu Val Leu Thr Pro Lys Ala 405 410 415Trp Asp
Arg Asn Gly Gln Asp Leu Thr Ala His Phe Thr Thr Ser Ile 420 425
430Pro Leu Lys Gly Asn Val Arg Asn Leu Ser Val Lys Ile Arg Glu Ala
435 440 445Thr Gly Leu Ala Trp Glu Trp Trp Arg Thr Val Tyr Glu Lys
Thr Asp 450 455 460Leu Pro Leu Val Arg Lys Arg Thr Ile Ser Ile Trp
Gly Thr Thr Leu465 470 475 480Tyr Pro Gln Val Glu Asp Lys Val Glu
Asn Asp 485 490301684DNAArtificial SequenceSynthetic polynucleotide
30tcaagctttt ggaccctcgt acagaagcta atacgactca ctatagggaa ataagagaga
60aaagaagagt aagaagaaat ataagagcca ccatggaaac ccctgcccag ctgctgttcc
120tgctgctgct gtggctgcct gacaccaccg gcatggccaa caaggccgtg
aacgacttca 180tcctggccat gaactacgac aagaagaagc tgctgaccca
ccagggcgag agcatcgaga 240acagattcat caaagagggc aaccagctgc
ccgacgagtt cgtcgtgatc gagcggaaga 300agcggagcct gagcaccaac
accagcgaca tcagcgtgac cgccaccaac gacagcagac 360tgtatcctgg
cgccctgctg gtggtggacg agacactgct ggaaaacaac cccaccctgc
420tggccgtgga cagagcccct atgacctaca gcatcgacct gcctggcctg
gccagcagcg 480atagctttct gcaggtggaa gatcccagca acagcagcgt
gcggggagcc gtgaatgacc 540tgctggctaa gtggcaccag gactacggcc
aagtgaacaa cgtgcccgcc agaatgcagt 600acgagaagat caccgcccac
tccatggaac agctgaaagt gaagttcggc agcgacttcg 660agaaaaccgg
caacagcctg gacatcgact tcaacagcgt gcacagcggc gagaagcaga
720tccagatcgt gaacttcaag cagatctact acaccgtgtc cgtgcgggcc
gtgaagaacc 780ctggggacgt gttccaggat accgtgaccg tggaagatct
gaagcagcgg ggcatcagcg 840ccgagaggcc actggtgtac atcagctctg
tggcctacgg cagacaggtg tacctgaagc 900tggaaaccac ctccaagagc
gacgaggtgg aagccgcctt cgaggccctg atcaagggcg 960tgaaagtggc
ccctcagacc gagtggaagc agattctgga caacaccgaa gtgaaagccg
1020tgatcctggg cggcgaccct tctagcggag ccagagtcgt gacaggcaag
gtggacatgg 1080tggaagatct gatccaggaa ggcagccggt tcaccgccga
tcaccctggc ctgcctatca 1140gctacaccac aagctttctg agagacaacg
tggtggccac attccagaac agcaccgact 1200acgtggaaac aaaagtgacc
gcctaccgga acggcgatct gctgctggat cactccggcg 1260cctacgtggc
ccagtactac atcacctggg acgagctgag ctacgatcac cagggcaaag
1320aggtgctgac ccccaaggcc tgggacagaa acggccagga tctgacagcc
cacttcacaa 1380ccagcatccc cctgaagggc aacgtgcgga acctgagcgt
gaagatcaga gagtgcaccg 1440gactggcctg ggagtggtgg cggaccgtgt
acgaaaagac cgacctgccc ctcgtgcgga 1500agcggaccat ctctatctgg
ggcaccacgc tgtatcctca ggtggaagat aaggtggaaa 1560acgactgata
ataggctgga gcctcggtgg ccatgcttct tgccccttgg gcctcccccc
1620agcccctcct ccccttcctg cacccgtacc cccgtggtct ttgaataaag
tctgagtggg 1680cggc 1684311684DNAArtificial SequenceSynthetic
polynucleotide 31tcaagctttt ggaccctcgt acagaagcta atacgactca
ctatagggaa ataagagaga 60aaagaagagt aagaagaaat ataagagcca ccatggaaac
ccctgcccag ctgctgttcc 120tgctgctgct gtggctgcct gacaccaccg
gcatggccaa caaggccgtg aacgacttca 180tcctggccat gaactacgac
aagaagaagc tgctgaccca ccagggcgag agcatcgaga 240acagattcat
caaagagggc aaccagctgc ccgacgagtt cgtcgtgatc gagcggaaga
300agcggagcct gagcaccaac accagcgaca tcagcgtgac cgccaccaac
gacagcagac 360tgtatcctgg cgccctgctg gtggtggacg agacactgct
ggaaaacaac cccaccctgc 420tggccgtgga cagagcccct atgacctaca
gcatcgacct gcctggcctg gccagcagcg 480atagctttct gcaggtggaa
gatcccagca acagcagcgt gcggggagcc gtgaatgacc 540tgctggctaa
gtggcaccag gactacggcc aagtgaacaa cgtgcccgcc agaatgcagt
600acgagaagat caccgcccac tccatggaac agctgaaagt gaagttcggc
agcgacttcg 660agaaaaccgg caacagcctg gacatcgact tcaacagcgt
gcacagcggc gagaagcaga 720tccagatcgt gaacttcaag cagatctact
acaccgtgtc cgtggacgcc gtgaagaacc 780ccggggacgt gttccaggat
accgtgaccg tggaagatct gaagcagcgg ggcatcagcg 840ccgagaggcc
actggtgtac atcagctctg tggcctacgg cagacaggtg tacctgaagc
900tggaaaccac ctccaagagc gacgaggtgg aagccgcctt cgaggccctg
atcaagggcg 960tgaaagtggc ccctcagacc gagtggaagc agattctgga
caacaccgaa gtgaaagccg 1020tgatcctggg cggcgaccct tctagcggag
ccagagtcgt gacaggcaag gtggacatgg 1080tggaagatct gatccaggaa
ggcagccggt tcaccgccga tcaccctggc ctgcctatca 1140gctacaccac
aagctttctg agagacaacg tggtggccac attccagaac agcaccgact
1200acgtggaaac aaaagtgacc gcctaccgga acggcgatct gctgctggat
cactccggcg 1260cctacgtggc ccagtactac atcacctggg acgagctgag
ctacgatcac cagggcaaag 1320aggtgctgac ccccaaggcc tgggacagaa
acggccagga tctgacagcc cacttcacaa 1380ccagcatccc cctgaagggc
aacgtgcgga acctgagcgt gaagatcaga gagtgcaccg 1440gactggcctg
ggagtggtgg cggaccgtgt acgaaaagac cgacctgccc ctcgtgcgga
1500agcggaccat ctctatctgg ggcaccaccg attaccccca ggtggaagat
aaggtggaaa 1560acgactgata ataggctgga gcctcggtgg ccatgcttct
tgccccttgg gcctcccccc 1620agcccctcct ccccttcctg cacccgtacc
cccgtggtct ttgaataaag tctgagtggg 1680cggc 1684321684DNAArtificial
SequenceSynthetic polynucleotide 32tcaagctttt ggaccctcgt acagaagcta
atacgactca ctatagggaa ataagagaga 60aaagaagagt aagaagaaat ataagagcca
ccatggaaac ccctgcccag ctgctgttcc 120tgctgctgct gtggctgcct
gacaccaccg gcatggccaa caaggccgtg aacgacttca 180tcctggccat
gaactacgac aagaagaagc tgctgaccca ccagggcgag agcatcgaga
240acagattcat caaagagggc aaccagctgc ccgacgagtt cgtcgtgatc
gagcggaaga 300agcggagcct gagcaccaac accagcgaca tcagcgtgac
cgcctgcaac gacagcagac 360tgtatcctgg cgccctgctg gtggtggacg
agacactgct ggaaaacaac cccaccctgc 420tggccgtgga cagagcccct
atgacctaca gcatcgacct gcctggcctg gccagcagcg 480atagctttct
gcaggtggaa gatcccagca acagcagcgt gcggggagcc gtgaatgacc
540tgctggctaa gtggcaccag gactacggcc aagtgaacaa cgtgcccgcc
agaatgcagt 600acgagaagat caccgcccac tccatggaac agctgaaagt
gaagttcggc agcgacttcg 660agaaaaccgg caacagcctg gacatcgact
tcaacagcgt gcacagcggc gagaagcaga 720tccagatcgt gaacttcaag
cagatctact acaccgtgtc cgtggacgcc gtgaagaacc 780ccggggacgt
gttccaggat accgtgaccg tggaagatct gaagcagcgg ggcatcagcg
840ccgagaggcc actggtgtac atcagctctg tggcctacgg cagacaggtg
tacctgaagc 900tggaaaccac ctccaagagc gacgaggtgg aagccgcctt
cgaggccctg atcaagggcg 960tgaaagtggc ccctcagacc gagtggaagc
agattctgga caacaccgaa gtgaaagccg 1020tgatcctgtg cggcgaccct
tctagcggag ccagagtcgt gacaggcaag gtggacatgg 1080tggaagatct
gatccaggaa ggcagccggt tcaccgccga tcaccctggc ctgcctatca
1140gctacaccac aagctttctg agagacaacg tggtggccac attccagaac
agcaccgact 1200acgtggaaac aaaagtgaca gcctaccgga acggcgatct
gctgctggat cactccggcg 1260cctacgtggc ccagtactac atcacctggg
acgagctgag ctacgatcac cagggcaaag 1320aggtgctgac ccccaaggcc
tgggacagaa acggccagga tctgacagcc cacttcacaa 1380ccagcatccc
cctgaagggc aacgtgcgga acctgagcgt gaagatcaga gaagccaccg
1440gactggcctg ggagtggtgg cggacagtgt acgaaaagac cgacctgccc
ctcgtgcgga 1500agcggaccat ctctatctgg ggcaccacgc tgtatcctca
ggtggaagat aaggtggaaa 1560acgactgata ataggctgga gcctcggtgg
ccatgcttct tgccccttgg gcctcccccc 1620agcccctcct ccccttcctg
cacccgtacc cccgtggtct ttgaataaag tctgagtggg 1680cggc
1684331684RNAArtificial SequenceSynthetic polynucleotide
33ucaagcuuuu ggacccucgu acagaagcua auacgacuca cuauagggaa auaagagaga
60aaagaagagu aagaagaaau auaagagcca ccauggaaac cccugcccag cugcuguucc
120ugcugcugcu guggcugccu gacaccaccg gcauggccaa caaggccgug
aacgacuuca 180uccuggccau gaacuacgac aagaagaagc ugcugaccca
ccagggcgag agcaucgaga 240acagauucau caaagagggc aaccagcugc
ccgacgaguu cgucgugauc gagcggaaga 300agcggagccu gagcaccaac
accagcgaca ucagcgugac cgccaccaac gacagcagac 360uguauccugg
cgcccugcug gugguggacg agacacugcu ggaaaacaac cccacccugc
420uggccgugga cagagccccu augaccuaca gcaucgaccu gccuggccug
gccagcagcg 480auagcuuucu gcagguggaa gaucccagca acagcagcgu
gcggggagcc gugaaugacc 540ugcuggcuaa guggcaccag gacuacggcc
aagugaacaa cgugcccgcc agaaugcagu 600acgagaagau caccgcccac
uccauggaac agcugaaagu gaaguucggc agcgacuucg 660agaaaaccgg
caacagccug gacaucgacu ucaacagcgu gcacagcggc gagaagcaga
720uccagaucgu gaacuucaag cagaucuacu acaccguguc cgugcgggcc
gugaagaacc 780cuggggacgu guuccaggau accgugaccg uggaagaucu
gaagcagcgg ggcaucagcg 840ccgagaggcc acugguguac aucagcucug
uggccuacgg cagacaggug uaccugaagc 900uggaaaccac cuccaagagc
gacgaggugg aagccgccuu cgaggcccug aucaagggcg 960ugaaaguggc
cccucagacc gaguggaagc agauucugga caacaccgaa gugaaagccg
1020ugauccuggg cggcgacccu ucuagcggag ccagagucgu gacaggcaag
guggacaugg 1080uggaagaucu gauccaggaa ggcagccggu ucaccgccga
ucacccuggc cugccuauca 1140gcuacaccac aagcuuucug agagacaacg
ugguggccac auuccagaac agcaccgacu 1200acguggaaac aaaagugacc
gccuaccgga acggcgaucu gcugcuggau cacuccggcg 1260ccuacguggc
ccaguacuac aucaccuggg acgagcugag cuacgaucac cagggcaaag
1320aggugcugac ccccaaggcc ugggacagaa acggccagga ucugacagcc
cacuucacaa 1380ccagcauccc ccugaagggc aacgugcgga accugagcgu
gaagaucaga gagugcaccg 1440gacuggccug ggaguggugg cggaccgugu
acgaaaagac cgaccugccc cucgugcgga 1500agcggaccau cucuaucugg
ggcaccacgc uguauccuca gguggaagau aagguggaaa 1560acgacugaua
auaggcugga gccucggugg ccaugcuucu ugccccuugg gccucccccc
1620agccccuccu ccccuuccug cacccguacc cccguggucu uugaauaaag
ucugaguggg 1680cggc 1684341684RNAArtificial SequenceSynthetic
polynucleotide 34ucaagcuuuu ggacccucgu acagaagcua auacgacuca
cuauagggaa auaagagaga 60aaagaagagu aagaagaaau auaagagcca ccauggaaac
cccugcccag cugcuguucc 120ugcugcugcu guggcugccu gacaccaccg
gcauggccaa caaggccgug aacgacuuca 180uccuggccau gaacuacgac
aagaagaagc ugcugaccca ccagggcgag agcaucgaga 240acagauucau
caaagagggc aaccagcugc ccgacgaguu cgucgugauc gagcggaaga
300agcggagccu gagcaccaac accagcgaca ucagcgugac cgccaccaac
gacagcagac 360uguauccugg cgcccugcug gugguggacg agacacugcu
ggaaaacaac cccacccugc 420uggccgugga cagagccccu augaccuaca
gcaucgaccu gccuggccug gccagcagcg 480auagcuuucu gcagguggaa
gaucccagca acagcagcgu gcggggagcc gugaaugacc 540ugcuggcuaa
guggcaccag gacuacggcc aagugaacaa cgugcccgcc agaaugcagu
600acgagaagau caccgcccac uccauggaac agcugaaagu gaaguucggc
agcgacuucg 660agaaaaccgg caacagccug gacaucgacu ucaacagcgu
gcacagcggc gagaagcaga 720uccagaucgu gaacuucaag cagaucuacu
acaccguguc cguggacgcc gugaagaacc 780ccggggacgu guuccaggau
accgugaccg uggaagaucu gaagcagcgg ggcaucagcg 840ccgagaggcc
acugguguac aucagcucug uggccuacgg cagacaggug uaccugaagc
900uggaaaccac cuccaagagc gacgaggugg aagccgccuu cgaggcccug
aucaagggcg 960ugaaaguggc cccucagacc gaguggaagc agauucugga
caacaccgaa gugaaagccg 1020ugauccuggg cggcgacccu ucuagcggag
ccagagucgu gacaggcaag guggacaugg 1080uggaagaucu gauccaggaa
ggcagccggu ucaccgccga ucacccuggc cugccuauca 1140gcuacaccac
aagcuuucug agagacaacg ugguggccac auuccagaac agcaccgacu
1200acguggaaac aaaagugacc gccuaccgga acggcgaucu gcugcuggau
cacuccggcg 1260ccuacguggc ccaguacuac aucaccuggg acgagcugag
cuacgaucac cagggcaaag 1320aggugcugac ccccaaggcc ugggacagaa
acggccagga ucugacagcc cacuucacaa 1380ccagcauccc ccugaagggc
aacgugcgga accugagcgu gaagaucaga gagugcaccg 1440gacuggccug
ggaguggugg cggaccgugu acgaaaagac cgaccugccc cucgugcgga
1500agcggaccau cucuaucugg ggcaccaccg auuaccccca gguggaagau
aagguggaaa 1560acgacugaua auaggcugga gccucggugg ccaugcuucu
ugccccuugg gccucccccc 1620agccccuccu ccccuuccug cacccguacc
cccguggucu uugaauaaag ucugaguggg 1680cggc 1684351684RNAArtificial
SequenceSynthetic polynucleotide 35ucaagcuuuu ggacccucgu acagaagcua
auacgacuca cuauagggaa auaagagaga 60aaagaagagu aagaagaaau auaagagcca
ccauggaaac cccugcccag cugcuguucc 120ugcugcugcu guggcugccu
gacaccaccg gcauggccaa caaggccgug aacgacuuca 180uccuggccau
gaacuacgac aagaagaagc ugcugaccca ccagggcgag agcaucgaga
240acagauucau caaagagggc aaccagcugc ccgacgaguu cgucgugauc
gagcggaaga 300agcggagccu gagcaccaac accagcgaca ucagcgugac
cgccugcaac gacagcagac 360uguauccugg cgcccugcug gugguggacg
agacacugcu ggaaaacaac cccacccugc 420uggccgugga cagagccccu
augaccuaca gcaucgaccu gccuggccug gccagcagcg 480auagcuuucu
gcagguggaa gaucccagca acagcagcgu gcggggagcc gugaaugacc
540ugcuggcuaa guggcaccag gacuacggcc aagugaacaa cgugcccgcc
agaaugcagu 600acgagaagau caccgcccac uccauggaac agcugaaagu
gaaguucggc agcgacuucg 660agaaaaccgg caacagccug gacaucgacu
ucaacagcgu gcacagcggc gagaagcaga 720uccagaucgu gaacuucaag
cagaucuacu acaccguguc cguggacgcc gugaagaacc 780ccggggacgu
guuccaggau accgugaccg uggaagaucu gaagcagcgg ggcaucagcg
840ccgagaggcc acugguguac aucagcucug uggccuacgg cagacaggug
uaccugaagc 900uggaaaccac cuccaagagc gacgaggugg aagccgccuu
cgaggcccug aucaagggcg 960ugaaaguggc cccucagacc gaguggaagc
agauucugga caacaccgaa gugaaagccg 1020ugauccugug cggcgacccu
ucuagcggag ccagagucgu gacaggcaag guggacaugg 1080uggaagaucu
gauccaggaa ggcagccggu ucaccgccga ucacccuggc cugccuauca
1140gcuacaccac aagcuuucug agagacaacg ugguggccac auuccagaac
agcaccgacu 1200acguggaaac aaaagugaca gccuaccgga acggcgaucu
gcugcuggau cacuccggcg 1260ccuacguggc ccaguacuac aucaccuggg
acgagcugag cuacgaucac cagggcaaag 1320aggugcugac ccccaaggcc
ugggacagaa acggccagga ucugacagcc cacuucacaa 1380ccagcauccc
ccugaagggc aacgugcgga accugagcgu gaagaucaga gaagccaccg
1440gacuggccug ggaguggugg cggacagugu acgaaaagac cgaccugccc
cucgugcgga 1500agcggaccau cucuaucugg ggcaccacgc uguauccuca
gguggaagau aagguggaaa 1560acgacugaua auaggcugga gccucggugg
ccaugcuucu ugccccuugg gccucccccc 1620agccccuccu ccccuuccug
cacccguacc cccguggucu uugaauaaag ucugaguggg 1680cggc
168436491PRTArtificial SequenceSynthetic polypeptide 36Met Glu Thr
Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro1 5 10 15Asp Thr
Thr Gly Met Ala Asn Lys Ala Val Asn Asp Phe Ile Leu Ala 20 25 30Met
Asn Tyr Asp Lys Lys Lys Leu Leu Thr His Gln Gly Glu Ser Ile 35 40
45Glu Asn Arg Phe Ile Lys Glu Gly Asn Gln Leu Pro Asp Glu Phe Val
50 55 60Val Ile Glu Arg Lys Lys Arg Ser Leu Ser Thr Asn Thr Ser Asp
Ile65 70 75 80Ser Val Thr Ala Thr Asn Asp Ser Arg Leu Tyr Pro Gly
Ala Leu Leu 85 90 95Val Val Asp Glu Thr Leu Leu Glu Asn Asn Pro Thr
Leu Leu Ala Val 100 105 110Asp Arg Ala Pro Met Thr Tyr Ser Ile Asp
Leu Pro Gly Leu Ala Ser 115 120 125Ser Asp Ser Phe Leu Gln Val Glu
Asp Pro Ser Asn Ser Ser Val Arg 130 135 140Gly Ala Val Asn Asp Leu
Leu Ala Lys Trp His Gln Asp Tyr Gly Gln145 150 155 160Val Asn Asn
Val Pro Ala Arg Met Gln Tyr Glu Lys Ile Thr Ala His 165 170 175Ser
Met Glu Gln Leu Lys Val Lys Phe Gly Ser Asp Phe Glu Lys Thr 180 185
190Gly Asn Ser Leu Asp Ile Asp Phe Asn Ser Val His Ser Gly Glu Lys
195 200 205Gln Ile Gln Ile Val Asn Phe Lys Gln Ile Tyr Tyr Thr Val
Ser Val 210 215 220Arg Ala Val Lys Asn Pro Gly Asp Val Phe Gln Asp
Thr Val Thr Val225 230 235 240Glu Asp Leu Lys Gln Arg Gly Ile Ser
Ala Glu Arg Pro Leu Val Tyr 245 250 255Ile Ser Ser Val Ala Tyr Gly
Arg Gln Val Tyr Leu Lys Leu Glu Thr 260 265 270Thr Ser Lys Ser Asp
Glu Val Glu Ala Ala Phe Glu Ala Leu Ile Lys 275 280 285Gly Val Lys
Val Ala Pro Gln Thr Glu Trp Lys Gln Ile Leu Asp Asn 290 295 300Thr
Glu Val Lys Ala Val Ile Leu Gly Gly Asp Pro Ser Ser Gly Ala305 310
315 320Arg Val Val Thr Gly Lys Val Asp Met Val Glu Asp Leu Ile Gln
Glu 325 330 335Gly Ser Arg Phe Thr Ala Asp His Pro Gly Leu Pro Ile
Ser Tyr Thr 340 345 350Thr Ser Phe Leu Arg Asp Asn Val Val Ala Thr
Phe Gln Asn Ser Thr 355 360 365Asp Tyr Val Glu Thr Lys Val Thr Ala
Tyr Arg Asn Gly Asp Leu Leu 370 375 380Leu Asp His Ser Gly Ala Tyr
Val Ala Gln Tyr Tyr Ile Thr Trp Asp385 390 395 400Glu Leu Ser Tyr
Asp His Gln Gly Lys Glu Val Leu Thr Pro Lys Ala 405 410 415Trp Asp
Arg Asn Gly Gln Asp Leu Thr Ala His Phe Thr Thr Ser Ile 420 425
430Pro Leu Lys Gly Asn Val Arg Asn Leu Ser Val Lys Ile Arg Glu Cys
435 440 445Thr Gly Leu Ala Trp Glu Trp Trp Arg Thr Val Tyr Glu Lys
Thr Asp 450 455 460Leu Pro Leu Val Arg Lys Arg Thr Ile Ser Ile Trp
Gly Thr Thr Leu465 470 475 480Tyr Pro Gln Val Glu Asp Lys Val Glu
Asn Asp 485 49037491PRTArtificial SequenceSynthetic polypeptide
37Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro1
5 10 15Asp Thr Thr Gly Met Ala Asn Lys Ala Val Asn Asp Phe Ile Leu
Ala 20 25 30Met Asn Tyr Asp Lys Lys Lys Leu Leu Thr His Gln Gly Glu
Ser Ile 35 40 45Glu Asn Arg Phe Ile Lys Glu Gly Asn Gln Leu Pro Asp
Glu Phe Val 50 55 60Val Ile Glu Arg Lys Lys Arg Ser Leu
Ser Thr Asn Thr Ser Asp Ile65 70 75 80Ser Val Thr Ala Thr Asn Asp
Ser Arg Leu Tyr Pro Gly Ala Leu Leu 85 90 95Val Val Asp Glu Thr Leu
Leu Glu Asn Asn Pro Thr Leu Leu Ala Val 100 105 110Asp Arg Ala Pro
Met Thr Tyr Ser Ile Asp Leu Pro Gly Leu Ala Ser 115 120 125Ser Asp
Ser Phe Leu Gln Val Glu Asp Pro Ser Asn Ser Ser Val Arg 130 135
140Gly Ala Val Asn Asp Leu Leu Ala Lys Trp His Gln Asp Tyr Gly
Gln145 150 155 160Val Asn Asn Val Pro Ala Arg Met Gln Tyr Glu Lys
Ile Thr Ala His 165 170 175Ser Met Glu Gln Leu Lys Val Lys Phe Gly
Ser Asp Phe Glu Lys Thr 180 185 190Gly Asn Ser Leu Asp Ile Asp Phe
Asn Ser Val His Ser Gly Glu Lys 195 200 205Gln Ile Gln Ile Val Asn
Phe Lys Gln Ile Tyr Tyr Thr Val Ser Val 210 215 220Asp Ala Val Lys
Asn Pro Gly Asp Val Phe Gln Asp Thr Val Thr Val225 230 235 240Glu
Asp Leu Lys Gln Arg Gly Ile Ser Ala Glu Arg Pro Leu Val Tyr 245 250
255Ile Ser Ser Val Ala Tyr Gly Arg Gln Val Tyr Leu Lys Leu Glu Thr
260 265 270Thr Ser Lys Ser Asp Glu Val Glu Ala Ala Phe Glu Ala Leu
Ile Lys 275 280 285Gly Val Lys Val Ala Pro Gln Thr Glu Trp Lys Gln
Ile Leu Asp Asn 290 295 300Thr Glu Val Lys Ala Val Ile Leu Gly Gly
Asp Pro Ser Ser Gly Ala305 310 315 320Arg Val Val Thr Gly Lys Val
Asp Met Val Glu Asp Leu Ile Gln Glu 325 330 335Gly Ser Arg Phe Thr
Ala Asp His Pro Gly Leu Pro Ile Ser Tyr Thr 340 345 350Thr Ser Phe
Leu Arg Asp Asn Val Val Ala Thr Phe Gln Asn Ser Thr 355 360 365Asp
Tyr Val Glu Thr Lys Val Thr Ala Tyr Arg Asn Gly Asp Leu Leu 370 375
380Leu Asp His Ser Gly Ala Tyr Val Ala Gln Tyr Tyr Ile Thr Trp
Asp385 390 395 400Glu Leu Ser Tyr Asp His Gln Gly Lys Glu Val Leu
Thr Pro Lys Ala 405 410 415Trp Asp Arg Asn Gly Gln Asp Leu Thr Ala
His Phe Thr Thr Ser Ile 420 425 430Pro Leu Lys Gly Asn Val Arg Asn
Leu Ser Val Lys Ile Arg Glu Cys 435 440 445Thr Gly Leu Ala Trp Glu
Trp Trp Arg Thr Val Tyr Glu Lys Thr Asp 450 455 460Leu Pro Leu Val
Arg Lys Arg Thr Ile Ser Ile Trp Gly Thr Thr Asp465 470 475 480Tyr
Pro Gln Val Glu Asp Lys Val Glu Asn Asp 485 49038491PRTArtificial
SequenceSynthetic polypeptide 38Met Glu Thr Pro Ala Gln Leu Leu Phe
Leu Leu Leu Leu Trp Leu Pro1 5 10 15Asp Thr Thr Gly Met Ala Asn Lys
Ala Val Asn Asp Phe Ile Leu Ala 20 25 30Met Asn Tyr Asp Lys Lys Lys
Leu Leu Thr His Gln Gly Glu Ser Ile 35 40 45Glu Asn Arg Phe Ile Lys
Glu Gly Asn Gln Leu Pro Asp Glu Phe Val 50 55 60Val Ile Glu Arg Lys
Lys Arg Ser Leu Ser Thr Asn Thr Ser Asp Ile65 70 75 80Ser Val Thr
Ala Cys Asn Asp Ser Arg Leu Tyr Pro Gly Ala Leu Leu 85 90 95Val Val
Asp Glu Thr Leu Leu Glu Asn Asn Pro Thr Leu Leu Ala Val 100 105
110Asp Arg Ala Pro Met Thr Tyr Ser Ile Asp Leu Pro Gly Leu Ala Ser
115 120 125Ser Asp Ser Phe Leu Gln Val Glu Asp Pro Ser Asn Ser Ser
Val Arg 130 135 140Gly Ala Val Asn Asp Leu Leu Ala Lys Trp His Gln
Asp Tyr Gly Gln145 150 155 160Val Asn Asn Val Pro Ala Arg Met Gln
Tyr Glu Lys Ile Thr Ala His 165 170 175Ser Met Glu Gln Leu Lys Val
Lys Phe Gly Ser Asp Phe Glu Lys Thr 180 185 190Gly Asn Ser Leu Asp
Ile Asp Phe Asn Ser Val His Ser Gly Glu Lys 195 200 205Gln Ile Gln
Ile Val Asn Phe Lys Gln Ile Tyr Tyr Thr Val Ser Val 210 215 220Asp
Ala Val Lys Asn Pro Gly Asp Val Phe Gln Asp Thr Val Thr Val225 230
235 240Glu Asp Leu Lys Gln Arg Gly Ile Ser Ala Glu Arg Pro Leu Val
Tyr 245 250 255Ile Ser Ser Val Ala Tyr Gly Arg Gln Val Tyr Leu Lys
Leu Glu Thr 260 265 270Thr Ser Lys Ser Asp Glu Val Glu Ala Ala Phe
Glu Ala Leu Ile Lys 275 280 285Gly Val Lys Val Ala Pro Gln Thr Glu
Trp Lys Gln Ile Leu Asp Asn 290 295 300Thr Glu Val Lys Ala Val Ile
Leu Cys Gly Asp Pro Ser Ser Gly Ala305 310 315 320Arg Val Val Thr
Gly Lys Val Asp Met Val Glu Asp Leu Ile Gln Glu 325 330 335Gly Ser
Arg Phe Thr Ala Asp His Pro Gly Leu Pro Ile Ser Tyr Thr 340 345
350Thr Ser Phe Leu Arg Asp Asn Val Val Ala Thr Phe Gln Asn Ser Thr
355 360 365Asp Tyr Val Glu Thr Lys Val Thr Ala Tyr Arg Asn Gly Asp
Leu Leu 370 375 380Leu Asp His Ser Gly Ala Tyr Val Ala Gln Tyr Tyr
Ile Thr Trp Asp385 390 395 400Glu Leu Ser Tyr Asp His Gln Gly Lys
Glu Val Leu Thr Pro Lys Ala 405 410 415Trp Asp Arg Asn Gly Gln Asp
Leu Thr Ala His Phe Thr Thr Ser Ile 420 425 430Pro Leu Lys Gly Asn
Val Arg Asn Leu Ser Val Lys Ile Arg Glu Ala 435 440 445Thr Gly Leu
Ala Trp Glu Trp Trp Arg Thr Val Tyr Glu Lys Thr Asp 450 455 460Leu
Pro Leu Val Arg Lys Arg Thr Ile Ser Ile Trp Gly Thr Thr Leu465 470
475 480Tyr Pro Gln Val Glu Asp Lys Val Glu Asn Asp 485
4903920PRTArtificial SequenceSynthetic polypeptide 39Met Glu Thr
Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro1 5 10 15Asp Thr
Thr Gly 204018PRTArtificial SequenceSynthetic polypeptide 40Met Asp
Trp Thr Trp Ile Leu Phe Leu Val Ala Ala Ala Thr Arg Val1 5 10 15His
Ser4124PRTJapanese encephalitis 41Met Leu Gly Ser Asn Ser Gly Gln
Arg Val Val Phe Thr Ile Leu Leu1 5 10 15Leu Leu Val Ala Pro Ala Tyr
Ser 204217PRTArtificial SequenceSynthetic polypeptide 42Met Lys Cys
Leu Leu Tyr Leu Ala Phe Leu Phe Ile Gly Val Asn Cys1 5 10
15Ala4315PRTJapanese encephalitis 43Met Trp Leu Val Ser Leu Ala Ile
Val Thr Ala Cys Ala Gly Ala1 5 10 15441729DNAArtificial
SequenceSynthetic polynucleotide 44tcaagctttt ggaccctcgt acagaagcta
atacgactca ctatagggaa ataagagaga 60aaagaagagt aagaagaaat ataagagcca
ccatggcaca agtcattaat acaaacagcc 120tgtcgctgtt gacccagaat
aacctgaaca aatcccagtc cgcactgggc actgctatcg 180agcgtttgtc
ttccggtctg cgtatcaaca gcgcgaaaga cgatgcggca ggacaggcga
240ttgctaaccg ttttaccgcg aacatcaaag gtctgactca ggcttcccgt
aacgctaacg 300acggtatctc cattgcgcag accactgaag gcgcgctgaa
cgaaatcaac aacaacctgc 360agcgtgtgcg tgaactggcg gttcagtctg
cgaatggtac taactcccag tctgacctcg 420actccatcca ggctgaaatc
acccagcgcc tgaacgaaat cgaccgtgta tccggccaga 480ctcagttcaa
cggcgtgaaa gtcctggcgc aggacaacac cctgaccatc caggttggtg
540ccaacgacgg tgaaactatc gatattgatt taaaagaaat cagctctaaa
acactgggac 600ttgataagct taatgtccaa gatgcctaca ccccgaaaga
aactgctgta accgttgata 660aaactaccta taaaaatggt acagatccta
ttacagccca gagcaatact gatatccaaa 720ctgcaattgg cggtggtgca
acgggggtta ctggggctga tatcaaattt aaagatggtc 780aatactattt
agatgttaaa ggcggtgctt ctgctggtgt ttataaagcc acttatgatg
840aaactacaaa gaaagttaat attgatacga ctgataaaac tccgttggca
actgcggaag 900ctacagctat tcggggaacg gccactataa cccacaacca
aattgctgaa gtaacaaaag 960agggtgttga tacgaccaca gttgcggctc
aacttgctgc agcaggggtt actggcgccg 1020ataaggacaa tactagcctt
gtaaaactat cgtttgagga taaaaacggt aaggttattg 1080atggtggcta
tgcagtgaaa atgggcgacg atttctatgc cgctacatat gatgagaaaa
1140caggtgcaat tactgctaaa accactactt atacagatgg tactggcgtt
gctcaaactg 1200gagctgtgaa atttggtggc gcaaatggta aatctgaagt
tgttactgct accgatggta 1260agacttactt agcaagcgac cttgacaaac
ataacttcag aacaggcggt gagcttaaag 1320aggttaatac agataagact
gaaaacccac tgcagaaaat tgatgctgcc ttggcacagg 1380ttgatacact
tcgttctgac ctgggtgcgg ttcagaaccg tttcaactcc gctatcacca
1440acctgggcaa taccgtaaat aacctgtctt ctgcccgtag ccgtatcgaa
gattccgact 1500acgcaaccga agtctccaac atgtctcgcg cgcagattct
gcagcaggcc ggtacctccg 1560ttctggcgca ggcgaaccag gttccgcaaa
acgtcctctc tttactgcgt tgataatagg 1620ctggagcctc ggtggccatg
cttcttgccc cttgggcctc cccccagccc ctcctcccct 1680tcctgcaccc
gtacccccgt ggtctttgaa taaagtctga gtgggcggc 1729451518DNAArtificial
SequenceSynthetic polynucleotide 45atggcacaag tcattaatac aaacagcctg
tcgctgttga cccagaataa cctgaacaaa 60tcccagtccg cactgggcac tgctatcgag
cgtttgtctt ccggtctgcg tatcaacagc 120gcgaaagacg atgcggcagg
acaggcgatt gctaaccgtt ttaccgcgaa catcaaaggt 180ctgactcagg
cttcccgtaa cgctaacgac ggtatctcca ttgcgcagac cactgaaggc
240gcgctgaacg aaatcaacaa caacctgcag cgtgtgcgtg aactggcggt
tcagtctgcg 300aatggtacta actcccagtc tgacctcgac tccatccagg
ctgaaatcac ccagcgcctg 360aacgaaatcg accgtgtatc cggccagact
cagttcaacg gcgtgaaagt cctggcgcag 420gacaacaccc tgaccatcca
ggttggtgcc aacgacggtg aaactatcga tattgattta 480aaagaaatca
gctctaaaac actgggactt gataagctta atgtccaaga tgcctacacc
540ccgaaagaaa ctgctgtaac cgttgataaa actacctata aaaatggtac
agatcctatt 600acagcccaga gcaatactga tatccaaact gcaattggcg
gtggtgcaac gggggttact 660ggggctgata tcaaatttaa agatggtcaa
tactatttag atgttaaagg cggtgcttct 720gctggtgttt ataaagccac
ttatgatgaa actacaaaga aagttaatat tgatacgact 780gataaaactc
cgttggcaac tgcggaagct acagctattc ggggaacggc cactataacc
840cacaaccaaa ttgctgaagt aacaaaagag ggtgttgata cgaccacagt
tgcggctcaa 900cttgctgcag caggggttac tggcgccgat aaggacaata
ctagccttgt aaaactatcg 960tttgaggata aaaacggtaa ggttattgat
ggtggctatg cagtgaaaat gggcgacgat 1020ttctatgccg ctacatatga
tgagaaaaca ggtgcaatta ctgctaaaac cactacttat 1080acagatggta
ctggcgttgc tcaaactgga gctgtgaaat ttggtggcgc aaatggtaaa
1140tctgaagttg ttactgctac cgatggtaag acttacttag caagcgacct
tgacaaacat 1200aacttcagaa caggcggtga gcttaaagag gttaatacag
ataagactga aaacccactg 1260cagaaaattg atgctgcctt ggcacaggtt
gatacacttc gttctgacct gggtgcggtt 1320cagaaccgtt tcaactccgc
tatcaccaac ctgggcaata ccgtaaataa cctgtcttct 1380gcccgtagcc
gtatcgaaga ttccgactac gcaaccgaag tctccaacat gtctcgcgcg
1440cagattctgc agcaggccgg tacctccgtt ctggcgcagg cgaaccaggt
tccgcaaaac 1500gtcctctctt tactgcgt 1518461790RNAArtificial
SequenceSynthetic polynucleotide 46ggggaaauaa gagagaaaag aagaguaaga
agaaauauaa gagccaccau ggcacaaguc 60auuaauacaa acagccuguc gcuguugacc
cagaauaacc ugaacaaauc ccaguccgca 120cugggcacug cuaucgagcg
uuugucuucc ggucugcgua ucaacagcgc gaaagacgau 180gcggcaggac
aggcgauugc uaaccguuuu accgcgaaca ucaaaggucu gacucaggcu
240ucccguaacg cuaacgacgg uaucuccauu gcgcagacca cugaaggcgc
gcugaacgaa 300aucaacaaca accugcagcg ugugcgugaa cuggcgguuc
agucugcgaa ugguacuaac 360ucccagucug accucgacuc cauccaggcu
gaaaucaccc agcgccugaa cgaaaucgac 420cguguauccg gccagacuca
guucaacggc gugaaagucc uggcgcagga caacacccug 480accauccagg
uuggugccaa cgacggugaa acuaucgaua uugauuuaaa agaaaucagc
540ucuaaaacac ugggacuuga uaagcuuaau guccaagaug ccuacacccc
gaaagaaacu 600gcuguaaccg uugauaaaac uaccuauaaa aaugguacag
auccuauuac agcccagagc 660aauacugaua uccaaacugc aauuggcggu
ggugcaacgg ggguuacugg ggcugauauc 720aaauuuaaag auggucaaua
cuauuuagau guuaaaggcg gugcuucugc ugguguuuau 780aaagccacuu
augaugaaac uacaaagaaa guuaauauug auacgacuga uaaaacuccg
840uuggcaacug cggaagcuac agcuauucgg ggaacggcca cuauaaccca
caaccaaauu 900gcugaaguaa caaaagaggg uguugauacg accacaguug
cggcucaacu ugcugcagca 960gggguuacug gcgccgauaa ggacaauacu
agccuuguaa aacuaucguu ugaggauaaa 1020aacgguaagg uuauugaugg
uggcuaugca gugaaaaugg gcgacgauuu cuaugccgcu 1080acauaugaug
agaaaacagg ugcaauuacu gcuaaaacca cuacuuauac agaugguacu
1140ggcguugcuc aaacuggagc ugugaaauuu gguggcgcaa augguaaauc
ugaaguuguu 1200acugcuaccg augguaagac uuacuuagca agcgaccuug
acaaacauaa cuucagaaca 1260ggcggugagc uuaaagaggu uaauacagau
aagacugaaa acccacugca gaaaauugau 1320gcugccuugg cacagguuga
uacacuucgu ucugaccugg gugcgguuca gaaccguuuc 1380aacuccgcua
ucaccaaccu gggcaauacc guaaauaacc ugucuucugc ccguagccgu
1440aucgaagauu ccgacuacgc aaccgaaguc uccaacaugu cucgcgcgca
gauucugcag 1500caggccggua ccuccguucu ggcgcaggcg aaccagguuc
cgcaaaacgu ccucucuuua 1560cugcguugau aauaggcugg agccucggug
gccaugcuuc uugccccuug ggccuccccc 1620cagccccucc uccccuuccu
gcacccguac ccccgugguc uuugaauaaa gucugagugg 1680gcggcaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1740aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaucuag
179047506PRTArtificial SequenceSynthetic polypeptide 47Met Ala Gln
Val Ile Asn Thr Asn Ser Leu Ser Leu Leu Thr Gln Asn1 5 10 15Asn Leu
Asn Lys Ser Gln Ser Ala Leu Gly Thr Ala Ile Glu Arg Leu 20 25 30Ser
Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln 35 40
45Ala Ile Ala Asn Arg Phe Thr Ala Asn Ile Lys Gly Leu Thr Gln Ala
50 55 60Ser Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu
Gly65 70 75 80Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val Arg
Glu Leu Ala 85 90 95Val Gln Ser Ala Asn Gly Thr Asn Ser Gln Ser Asp
Leu Asp Ser Ile 100 105 110Gln Ala Glu Ile Thr Gln Arg Leu Asn Glu
Ile Asp Arg Val Ser Gly 115 120 125Gln Thr Gln Phe Asn Gly Val Lys
Val Leu Ala Gln Asp Asn Thr Leu 130 135 140Thr Ile Gln Val Gly Ala
Asn Asp Gly Glu Thr Ile Asp Ile Asp Leu145 150 155 160Lys Glu Ile
Ser Ser Lys Thr Leu Gly Leu Asp Lys Leu Asn Val Gln 165 170 175Asp
Ala Tyr Thr Pro Lys Glu Thr Ala Val Thr Val Asp Lys Thr Thr 180 185
190Tyr Lys Asn Gly Thr Asp Pro Ile Thr Ala Gln Ser Asn Thr Asp Ile
195 200 205Gln Thr Ala Ile Gly Gly Gly Ala Thr Gly Val Thr Gly Ala
Asp Ile 210 215 220Lys Phe Lys Asp Gly Gln Tyr Tyr Leu Asp Val Lys
Gly Gly Ala Ser225 230 235 240Ala Gly Val Tyr Lys Ala Thr Tyr Asp
Glu Thr Thr Lys Lys Val Asn 245 250 255Ile Asp Thr Thr Asp Lys Thr
Pro Leu Ala Thr Ala Glu Ala Thr Ala 260 265 270Ile Arg Gly Thr Ala
Thr Ile Thr His Asn Gln Ile Ala Glu Val Thr 275 280 285Lys Glu Gly
Val Asp Thr Thr Thr Val Ala Ala Gln Leu Ala Ala Ala 290 295 300Gly
Val Thr Gly Ala Asp Lys Asp Asn Thr Ser Leu Val Lys Leu Ser305 310
315 320Phe Glu Asp Lys Asn Gly Lys Val Ile Asp Gly Gly Tyr Ala Val
Lys 325 330 335Met Gly Asp Asp Phe Tyr Ala Ala Thr Tyr Asp Glu Lys
Thr Gly Ala 340 345 350Ile Thr Ala Lys Thr Thr Thr Tyr Thr Asp Gly
Thr Gly Val Ala Gln 355 360 365Thr Gly Ala Val Lys Phe Gly Gly Ala
Asn Gly Lys Ser Glu Val Val 370 375 380Thr Ala Thr Asp Gly Lys Thr
Tyr Leu Ala Ser Asp Leu Asp Lys His385 390 395 400Asn Phe Arg Thr
Gly Gly Glu Leu Lys Glu Val Asn Thr Asp Lys Thr 405 410 415Glu Asn
Pro Leu Gln Lys Ile Asp Ala Ala Leu Ala Gln Val Asp Thr 420 425
430Leu Arg Ser Asp Leu Gly Ala Val Gln Asn Arg Phe Asn Ser Ala Ile
435 440 445Thr Asn Leu Gly Asn Thr Val Asn Asn Leu Ser Ser Ala Arg
Ser Arg 450 455 460Ile Glu Asp Ser Asp Tyr Ala Thr Glu Val Ser Asn
Met Ser Arg Ala465 470 475 480Gln Ile Leu Gln Gln Ala Gly Thr Ser
Val Leu Ala Gln Ala Asn Gln 485 490 495Val Pro Gln Asn Val Leu Ser
Leu Leu Arg 500 50548698PRTArtificial SequenceSynthetic polypeptide
48Met Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Leu Thr Gln Asn1
5 10 15Asn Leu Asn Lys Ser Gln Ser Ala Leu Gly Thr Ala Ile Glu Arg
Leu 20 25 30Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala
Gly Gln
35 40 45Ala Ile Ala Asn Arg Phe Thr Ala Asn Ile Lys Gly Leu Thr Gln
Ala 50 55 60Ser Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr
Glu Gly65 70 75 80Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val
Arg Glu Leu Ala 85 90 95Val Gln Ser Ala Asn Ser Thr Asn Ser Gln Ser
Asp Leu Asp Ser Ile 100 105 110Gln Ala Glu Ile Thr Gln Arg Leu Asn
Glu Ile Asp Arg Val Ser Gly 115 120 125Gln Thr Gln Phe Asn Gly Val
Lys Val Leu Ala Gln Asp Asn Thr Leu 130 135 140Thr Ile Gln Val Gly
Ala Asn Asp Gly Glu Thr Ile Asp Ile Asp Leu145 150 155 160Lys Gln
Ile Asn Ser Gln Thr Leu Gly Leu Asp Thr Leu Asn Val Gln 165 170
175Gln Lys Tyr Lys Val Ser Asp Thr Ala Ala Thr Val Thr Gly Tyr Ala
180 185 190Asp Thr Thr Ile Ala Leu Asp Asn Ser Thr Phe Lys Ala Ser
Ala Thr 195 200 205Gly Leu Gly Gly Thr Asp Gln Lys Ile Asp Gly Asp
Leu Lys Phe Asp 210 215 220Asp Thr Thr Gly Lys Tyr Tyr Ala Lys Val
Thr Val Thr Gly Gly Thr225 230 235 240Gly Lys Asp Gly Tyr Tyr Glu
Val Ser Val Asp Lys Thr Asn Gly Glu 245 250 255Val Thr Leu Ala Gly
Gly Ala Thr Ser Pro Leu Thr Gly Gly Leu Pro 260 265 270Ala Thr Ala
Thr Glu Asp Val Lys Asn Val Gln Val Ala Asn Ala Asp 275 280 285Leu
Thr Glu Ala Lys Ala Ala Leu Thr Ala Ala Gly Val Thr Gly Thr 290 295
300Ala Ser Val Val Lys Met Ser Tyr Thr Asp Asn Asn Gly Lys Thr
Ile305 310 315 320Asp Gly Gly Leu Ala Val Lys Val Gly Asp Asp Tyr
Tyr Ser Ala Thr 325 330 335Gln Asn Lys Asp Gly Ser Ile Ser Ile Asn
Thr Thr Lys Tyr Thr Ala 340 345 350Asp Asp Gly Thr Ser Lys Thr Ala
Leu Asn Lys Leu Gly Gly Ala Asp 355 360 365Gly Lys Thr Glu Val Val
Ser Ile Gly Gly Lys Thr Tyr Ala Ala Ser 370 375 380Lys Ala Glu Gly
His Asn Phe Lys Ala Gln Pro Asp Leu Ala Glu Ala385 390 395 400Ala
Ala Thr Thr Thr Glu Asn Pro Leu Gln Lys Ile Asp Ala Ala Leu 405 410
415Ala Gln Val Asp Thr Leu Arg Ser Asp Leu Gly Ala Val Gln Asn Arg
420 425 430Phe Asn Ser Ala Ile Thr Asn Leu Gly Asn Thr Val Asn Asn
Leu Thr 435 440 445Ser Ala Arg Ser Arg Ile Glu Asp Ser Asp Tyr Ala
Thr Glu Val Ser 450 455 460Asn Met Ser Arg Ala Gln Ile Leu Gln Gln
Ala Gly Thr Ser Val Leu465 470 475 480Ala Gln Ala Asn Gln Val Pro
Gln Asn Val Leu Ser Leu Leu Arg Gly 485 490 495Gly Gly Gly Ser Gly
Gly Gly Gly Ser Met Met Ala Pro Asp Pro Asn 500 505 510Ala Asn Pro
Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 515 520 525Ala
Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn 530 535
540Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro
Asn545 550 555 560Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn
Ala Asn Pro Asn 565 570 575Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn
Pro Asn Lys Asn Asn Gln 580 585 590Gly Asn Gly Gln Gly His Asn Met
Pro Asn Asp Pro Asn Arg Asn Val 595 600 605Asp Glu Asn Ala Asn Ala
Asn Asn Ala Val Lys Asn Asn Asn Asn Glu 610 615 620Glu Pro Ser Asp
Lys His Ile Glu Gln Tyr Leu Lys Lys Ile Lys Asn625 630 635 640Ser
Ile Ser Thr Glu Trp Ser Pro Cys Ser Val Thr Cys Gly Asn Gly 645 650
655Ile Gln Val Arg Ile Lys Pro Gly Ser Ala Asn Lys Pro Lys Asp Glu
660 665 670Leu Asp Tyr Glu Asn Asp Ile Glu Lys Lys Ile Cys Lys Met
Glu Lys 675 680 685Cys Ser Ser Val Phe Asn Val Val Asn Ser 690
69549692PRTArtificial SequenceSynthetic polypeptide 49Met Met Ala
Pro Asp Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala1 5 10 15Asn Pro
Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala 20 25 30Asn
Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala 35 40
45Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala
50 55 60Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn Ala Asn Pro Asn
Ala65 70 75 80Asn Pro Asn Lys Asn Asn Gln Gly Asn Gly Gln Gly His
Asn Met Pro 85 90 95Asn Asp Pro Asn Arg Asn Val Asp Glu Asn Ala Asn
Ala Asn Asn Ala 100 105 110Val Lys Asn Asn Asn Asn Glu Glu Pro Ser
Asp Lys His Ile Glu Gln 115 120 125Tyr Leu Lys Lys Ile Lys Asn Ser
Ile Ser Thr Glu Trp Ser Pro Cys 130 135 140Ser Val Thr Cys Gly Asn
Gly Ile Gln Val Arg Ile Lys Pro Gly Ser145 150 155 160Ala Asn Lys
Pro Lys Asp Glu Leu Asp Tyr Glu Asn Asp Ile Glu Lys 165 170 175Lys
Ile Cys Lys Met Glu Lys Cys Ser Ser Val Phe Asn Val Val Asn 180 185
190Ser Arg Pro Val Thr Met Ala Gln Val Ile Asn Thr Asn Ser Leu Ser
195 200 205Leu Leu Thr Gln Asn Asn Leu Asn Lys Ser Gln Ser Ala Leu
Gly Thr 210 215 220Ala Ile Glu Arg Leu Ser Ser Gly Leu Arg Ile Asn
Ser Ala Lys Asp225 230 235 240Asp Ala Ala Gly Gln Ala Ile Ala Asn
Arg Phe Thr Ala Asn Ile Lys 245 250 255Gly Leu Thr Gln Ala Ser Arg
Asn Ala Asn Asp Gly Ile Ser Ile Ala 260 265 270Gln Thr Thr Glu Gly
Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg 275 280 285Val Arg Glu
Leu Ala Val Gln Ser Ala Asn Ser Thr Asn Ser Gln Ser 290 295 300Asp
Leu Asp Ser Ile Gln Ala Glu Ile Thr Gln Arg Leu Asn Glu Ile305 310
315 320Asp Arg Val Ser Gly Gln Thr Gln Phe Asn Gly Val Lys Val Leu
Ala 325 330 335Gln Asp Asn Thr Leu Thr Ile Gln Val Gly Ala Asn Asp
Gly Glu Thr 340 345 350Ile Asp Ile Asp Leu Lys Gln Ile Asn Ser Gln
Thr Leu Gly Leu Asp 355 360 365Thr Leu Asn Val Gln Gln Lys Tyr Lys
Val Ser Asp Thr Ala Ala Thr 370 375 380Val Thr Gly Tyr Ala Asp Thr
Thr Ile Ala Leu Asp Asn Ser Thr Phe385 390 395 400Lys Ala Ser Ala
Thr Gly Leu Gly Gly Thr Asp Gln Lys Ile Asp Gly 405 410 415Asp Leu
Lys Phe Asp Asp Thr Thr Gly Lys Tyr Tyr Ala Lys Val Thr 420 425
430Val Thr Gly Gly Thr Gly Lys Asp Gly Tyr Tyr Glu Val Ser Val Asp
435 440 445Lys Thr Asn Gly Glu Val Thr Leu Ala Gly Gly Ala Thr Ser
Pro Leu 450 455 460Thr Gly Gly Leu Pro Ala Thr Ala Thr Glu Asp Val
Lys Asn Val Gln465 470 475 480Val Ala Asn Ala Asp Leu Thr Glu Ala
Lys Ala Ala Leu Thr Ala Ala 485 490 495Gly Val Thr Gly Thr Ala Ser
Val Val Lys Met Ser Tyr Thr Asp Asn 500 505 510Asn Gly Lys Thr Ile
Asp Gly Gly Leu Ala Val Lys Val Gly Asp Asp 515 520 525Tyr Tyr Ser
Ala Thr Gln Asn Lys Asp Gly Ser Ile Ser Ile Asn Thr 530 535 540Thr
Lys Tyr Thr Ala Asp Asp Gly Thr Ser Lys Thr Ala Leu Asn Lys545 550
555 560Leu Gly Gly Ala Asp Gly Lys Thr Glu Val Val Ser Ile Gly Gly
Lys 565 570 575Thr Tyr Ala Ala Ser Lys Ala Glu Gly His Asn Phe Lys
Ala Gln Pro 580 585 590Asp Leu Ala Glu Ala Ala Ala Thr Thr Thr Glu
Asn Pro Leu Gln Lys 595 600 605Ile Asp Ala Ala Leu Ala Gln Val Asp
Thr Leu Arg Ser Asp Leu Gly 610 615 620Ala Val Gln Asn Arg Phe Asn
Ser Ala Ile Thr Asn Leu Gly Asn Thr625 630 635 640Val Asn Asn Leu
Thr Ser Ala Arg Ser Arg Ile Glu Asp Ser Asp Tyr 645 650 655Ala Thr
Glu Val Ser Asn Met Ser Arg Ala Gln Ile Leu Gln Gln Ala 660 665
670Gly Thr Ser Val Leu Ala Gln Ala Asn Gln Val Pro Gln Asn Val Leu
675 680 685Ser Leu Leu Arg 690501729RNAArtificial SequenceSynthetic
polynucleotide 50ucaagcuuuu ggacccucgu acagaagcua auacgacuca
cuauagggaa auaagagaga 60aaagaagagu aagaagaaau auaagagcca ccauggcaca
agucauuaau acaaacagcc 120ugucgcuguu gacccagaau aaccugaaca
aaucccaguc cgcacugggc acugcuaucg 180agcguuuguc uuccggucug
cguaucaaca gcgcgaaaga cgaugcggca ggacaggcga 240uugcuaaccg
uuuuaccgcg aacaucaaag gucugacuca ggcuucccgu aacgcuaacg
300acgguaucuc cauugcgcag accacugaag gcgcgcugaa cgaaaucaac
aacaaccugc 360agcgugugcg ugaacuggcg guucagucug cgaaugguac
uaacucccag ucugaccucg 420acuccaucca ggcugaaauc acccagcgcc
ugaacgaaau cgaccgugua uccggccaga 480cucaguucaa cggcgugaaa
guccuggcgc aggacaacac ccugaccauc cagguuggug 540ccaacgacgg
ugaaacuauc gauauugauu uaaaagaaau cagcucuaaa acacugggac
600uugauaagcu uaauguccaa gaugccuaca ccccgaaaga aacugcugua
accguugaua 660aaacuaccua uaaaaauggu acagauccua uuacagccca
gagcaauacu gauauccaaa 720cugcaauugg cgguggugca acggggguua
cuggggcuga uaucaaauuu aaagaugguc 780aauacuauuu agauguuaaa
ggcggugcuu cugcuggugu uuauaaagcc acuuaugaug 840aaacuacaaa
gaaaguuaau auugauacga cugauaaaac uccguuggca acugcggaag
900cuacagcuau ucggggaacg gccacuauaa cccacaacca aauugcugaa
guaacaaaag 960aggguguuga uacgaccaca guugcggcuc aacuugcugc
agcagggguu acuggcgccg 1020auaaggacaa uacuagccuu guaaaacuau
cguuugagga uaaaaacggu aagguuauug 1080augguggcua ugcagugaaa
augggcgacg auuucuaugc cgcuacauau gaugagaaaa 1140caggugcaau
uacugcuaaa accacuacuu auacagaugg uacuggcguu gcucaaacug
1200gagcugugaa auuugguggc gcaaauggua aaucugaagu uguuacugcu
accgauggua 1260agacuuacuu agcaagcgac cuugacaaac auaacuucag
aacaggcggu gagcuuaaag 1320agguuaauac agauaagacu gaaaacccac
ugcagaaaau ugaugcugcc uuggcacagg 1380uugauacacu ucguucugac
cugggugcgg uucagaaccg uuucaacucc gcuaucacca 1440accugggcaa
uaccguaaau aaccugucuu cugcccguag ccguaucgaa gauuccgacu
1500acgcaaccga agucuccaac augucucgcg cgcagauucu gcagcaggcc
gguaccuccg 1560uucuggcgca ggcgaaccag guuccgcaaa acguccucuc
uuuacugcgu ugauaauagg 1620cuggagccuc gguggccaug cuucuugccc
cuugggccuc cccccagccc cuccuccccu 1680uccugcaccc guacccccgu
ggucuuugaa uaaagucuga gugggcggc 1729511518RNAArtificial
SequenceSynthetic polynucleotide 51auggcacaag ucauuaauac aaacagccug
ucgcuguuga cccagaauaa ccugaacaaa 60ucccaguccg cacugggcac ugcuaucgag
cguuugucuu ccggucugcg uaucaacagc 120gcgaaagacg augcggcagg
acaggcgauu gcuaaccguu uuaccgcgaa caucaaaggu 180cugacucagg
cuucccguaa cgcuaacgac gguaucucca uugcgcagac cacugaaggc
240gcgcugaacg aaaucaacaa caaccugcag cgugugcgug aacuggcggu
ucagucugcg 300aaugguacua acucccaguc ugaccucgac uccauccagg
cugaaaucac ccagcgccug 360aacgaaaucg accguguauc cggccagacu
caguucaacg gcgugaaagu ccuggcgcag 420gacaacaccc ugaccaucca
gguuggugcc aacgacggug aaacuaucga uauugauuua 480aaagaaauca
gcucuaaaac acugggacuu gauaagcuua auguccaaga ugccuacacc
540ccgaaagaaa cugcuguaac cguugauaaa acuaccuaua aaaaugguac
agauccuauu 600acagcccaga gcaauacuga uauccaaacu gcaauuggcg
guggugcaac ggggguuacu 660ggggcugaua ucaaauuuaa agauggucaa
uacuauuuag auguuaaagg cggugcuucu 720gcugguguuu auaaagccac
uuaugaugaa acuacaaaga aaguuaauau ugauacgacu 780gauaaaacuc
cguuggcaac ugcggaagcu acagcuauuc ggggaacggc cacuauaacc
840cacaaccaaa uugcugaagu aacaaaagag gguguugaua cgaccacagu
ugcggcucaa 900cuugcugcag cagggguuac uggcgccgau aaggacaaua
cuagccuugu aaaacuaucg 960uuugaggaua aaaacgguaa gguuauugau
gguggcuaug cagugaaaau gggcgacgau 1020uucuaugccg cuacauauga
ugagaaaaca ggugcaauua cugcuaaaac cacuacuuau 1080acagauggua
cuggcguugc ucaaacugga gcugugaaau uugguggcgc aaaugguaaa
1140ucugaaguug uuacugcuac cgaugguaag acuuacuuag caagcgaccu
ugacaaacau 1200aacuucagaa caggcgguga gcuuaaagag guuaauacag
auaagacuga aaacccacug 1260cagaaaauug augcugccuu ggcacagguu
gauacacuuc guucugaccu gggugcgguu 1320cagaaccguu ucaacuccgc
uaucaccaac cugggcaaua ccguaaauaa ccugucuucu 1380gcccguagcc
guaucgaaga uuccgacuac gcaaccgaag ucuccaacau gucucgcgcg
1440cagauucugc agcaggccgg uaccuccguu cuggcgcagg cgaaccaggu
uccgcaaaac 1500guccucucuu uacugcgu 1518521790RNAArtificial
SequenceSynthetic polynucleotide 52ggggaaauaa gagagaaaag aagaguaaga
agaaauauaa gagccaccau ggcacaaguc 60auuaauacaa acagccuguc gcuguugacc
cagaauaacc ugaacaaauc ccaguccgca 120cugggcacug cuaucgagcg
uuugucuucc ggucugcgua ucaacagcgc gaaagacgau 180gcggcaggac
aggcgauugc uaaccguuuu accgcgaaca ucaaaggucu gacucaggcu
240ucccguaacg cuaacgacgg uaucuccauu gcgcagacca cugaaggcgc
gcugaacgaa 300aucaacaaca accugcagcg ugugcgugaa cuggcgguuc
agucugcgaa ugguacuaac 360ucccagucug accucgacuc cauccaggcu
gaaaucaccc agcgccugaa cgaaaucgac 420cguguauccg gccagacuca
guucaacggc gugaaagucc uggcgcagga caacacccug 480accauccagg
uuggugccaa cgacggugaa acuaucgaua uugauuuaaa agaaaucagc
540ucuaaaacac ugggacuuga uaagcuuaau guccaagaug ccuacacccc
gaaagaaacu 600gcuguaaccg uugauaaaac uaccuauaaa aaugguacag
auccuauuac agcccagagc 660aauacugaua uccaaacugc aauuggcggu
ggugcaacgg ggguuacugg ggcugauauc 720aaauuuaaag auggucaaua
cuauuuagau guuaaaggcg gugcuucugc ugguguuuau 780aaagccacuu
augaugaaac uacaaagaaa guuaauauug auacgacuga uaaaacuccg
840uuggcaacug cggaagcuac agcuauucgg ggaacggcca cuauaaccca
caaccaaauu 900gcugaaguaa caaaagaggg uguugauacg accacaguug
cggcucaacu ugcugcagca 960gggguuacug gcgccgauaa ggacaauacu
agccuuguaa aacuaucguu ugaggauaaa 1020aacgguaagg uuauugaugg
uggcuaugca gugaaaaugg gcgacgauuu cuaugccgcu 1080acauaugaug
agaaaacagg ugcaauuacu gcuaaaacca cuacuuauac agaugguacu
1140ggcguugcuc aaacuggagc ugugaaauuu gguggcgcaa augguaaauc
ugaaguuguu 1200acugcuaccg augguaagac uuacuuagca agcgaccuug
acaaacauaa cuucagaaca 1260ggcggugagc uuaaagaggu uaauacagau
aagacugaaa acccacugca gaaaauugau 1320gcugccuugg cacagguuga
uacacuucgu ucugaccugg gugcgguuca gaaccguuuc 1380aacuccgcua
ucaccaaccu gggcaauacc guaaauaacc ugucuucugc ccguagccgu
1440aucgaagauu ccgacuacgc aaccgaaguc uccaacaugu cucgcgcgca
gauucugcag 1500caggccggua ccuccguucu ggcgcaggcg aaccagguuc
cgcaaaacgu ccucucuuua 1560cugcguugau aauaggcugg agccucggug
gccaugcuuc uugccccuug ggccuccccc 1620cagccccucc uccccuuccu
gcacccguac ccccgugguc uuugaauaaa gucugagugg 1680gcggcaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1740aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaucuag
17905313PRTArtificial SequenceSynthetic polypeptide 53Leu Gln Arg
Val Arg Glu Leu Ala Val Gln Ser Ala Asn1 5 105447RNAArtificial
SequenceSynthetic polynycleotide 54gggaaauaag agagaaaaga agaguaagaa
gaaauauaag agccacc 4755119RNAArtificial SequenceSynthetic
polynucleotide 55ugauaauagg cuggagccuc gguggccuag cuucuugccc
cuugggccuc cccccagccc 60cuccuccccu uccugcaccc guacccccgu ggucuuugaa
uaaagucuga gugggcggc 119561473DNAArtificial SequenceSynthetic
polynucleotide 56atggaaaccc ctgcccagct gctgttcctg ctgctgctgt
ggctgcctga caccaccggc 60atggccaaca aggccgtgaa cgacttcatc ctggccatga
actacgacaa gaagaagctg 120ctgacccacc agggcgagag catcgagaac
agattcatca aagagggcaa ccagctgccc 180gacgagttcg tcgtgatcga
gcggaagaag cggagcctga gcaccgacac cagcgacatc 240agcgtgaccg
ccaccaacga cgccagactg tatcctggcg ctctgctggt ggtggacgag
300acactgctgg aaaacaaccc catcctgctg gccgtggaca gagcccccat
gacctacagc 360atcgacctgc ctggcctggc cagcagcgat agctttctgc
aggtggaaga tcccagcaac 420agcgccgtgc ggggagccgt gaatgacctg
ctggctaagt ggcaccagga ctacggccaa 480gtgaacaacg tgcccgccag
aatgcagtac gagaagatca ccgcccactc catggaacag 540ctgaaagtga
agttcggcag cgacttcgag aaaaccggca acagcctgga catcgacttc
600aacagcgtgc acagcggcga gaagcagatc cagatcgtga acttcaagca
gatctactac 660accgtgtccg tgcgggccgt gaagaaccct ggggacgtgt
tccaggatac cgtgaccgtg 720gaagatctga agcagcgggg catcagcgcc
gagaggccac tggtgtacat cagcagcgtg 780gcctacggca gacaggtgta
cctgaagctg gaaaccacct ccaagagcga cgaggtggaa 840gccgccttcg
aggccctgat caagggcgtg aaagtggccc ctcagaccga gtggaagcag
900attctggaca acaccgaagt gaaagccgtg atcctgggcg gcgacccttc
tagcggagcc 960agagtcgtga caggcaaggt ggacatggtg gaagatctga
tccaggaagg cagccggttc 1020accgccgatc accctggcct gcctatcagc
tacaccacaa gctttctgag agacaacgtg 1080gtggccacat
tccagaactc cgccgactac gtggaaacaa aagtgaccgc ctaccggaac
1140ggcgatctgc tgctggatca ctccggcgcc tatgtggccc agtactacat
cacctgggac 1200gagctgagct acgatcacca gggcaaagag gtgctgaccc
ccaaggcctg ggacagaaac 1260ggccaggatc tgacagccca cttcacaacc
agcatccccc tgaagggcaa cgtgcggaac 1320ctgagcgtga agatcagaga
gtgcaccgga ctggcctggg agtggtggcg gaccgtgtac 1380gaaaagaccg
acctgcccct cgtgcggaag cggaccatct ctatctgggg caccacgctg
1440tatcctcagg tggaagataa ggtggaaaac gac 1473571473DNAArtificial
SequenceSynthetic polynucleotide 57atggaaaccc ctgcccagct gctgttcctg
ctgctgctgt ggctgcctga caccaccggc 60atggccaaca aggccgtgaa cgacttcatc
ctggccatga actacgacaa gaagaagctg 120ctgacccacc agggcgagag
catcgagaac agattcatca aagagggcaa ccagctgccc 180gacgagttcg
tcgtgatcga gcggaagaag cggagcctga gcaccgacac cagcgacatc
240agcgtgaccg ccaccaacga cgccagactg tatcctggcg ctctgctggt
ggtggacgag 300acactgctgg aaaacaaccc catcctgctg gccgtggaca
gagcccccat gacctacagc 360atcgacctgc ctggcctggc cagcagcgat
agctttctgc aggtggaaga tcccagcaac 420agcgccgtgc ggggagccgt
gaatgacctg ctggctaagt ggcaccagga ctacggccaa 480gtgaacaacg
tgcccgccag aatgcagtac gagaagatca ccgcccactc catggaacag
540ctgaaagtga agttcggcag cgacttcgag aaaaccggca acagcctgga
catcgacttc 600aacagcgtgc acagcggcga gaagcagatc cagatcgtga
acttcaagca gatctactac 660accgtgtccg tggacgccgt gaagaacccc
ggggacgtgt tccaggatac cgtgaccgtg 720gaagatctga agcagcgggg
catcagcgcc gagaggccac tggtgtacat cagcagcgtg 780gcctacggca
gacaggtgta cctgaagctg gaaaccacct ccaagagcga cgaggtggaa
840gccgccttcg aggccctgat caagggcgtg aaagtggccc ctcagaccga
gtggaagcag 900attctggaca acaccgaagt gaaagccgtg atcctgggcg
gcgacccttc tagcggagcc 960agagtcgtga caggcaaggt ggacatggtg
gaagatctga tccaggaagg cagccggttc 1020accgccgatc accctggcct
gcctatcagc tacaccacaa gctttctgag agacaacgtg 1080gtggccacat
tccagaactc cgccgactac gtggaaacaa aagtgaccgc ctaccggaac
1140ggcgatctgc tgctggatca ctccggcgcc tatgtggccc agtactacat
cacctgggac 1200gagctgagct acgatcacca gggcaaagag gtgctgaccc
ccaaggcctg ggacagaaac 1260ggccaggatc tgacagccca cttcacaacc
agcatccccc tgaagggcaa cgtgcggaac 1320ctgagcgtga agatcagaga
gtgcaccgga ctggcctggg agtggtggcg gaccgtgtac 1380gaaaagaccg
acctgcccct cgtgcggaag cggaccatct ctatctgggg caccaccgac
1440tacccccagg tggaagataa ggtggaaaac gac 1473581473DNAArtificial
SequenceSynthetic polynucleotide 58atggaaaccc ctgcccagct gctgttcctg
ctgctgctgt ggctgcctga caccaccggc 60atggccaaca aggccgtgaa cgacttcatc
ctggccatga actacgacaa gaagaagctg 120ctgacccacc agggcgagag
catcgagaac agattcatca aagagggcaa ccagctgccc 180gacgagttcg
tcgtgatcga gcggaagaag cggagcctga gcaccgacac cagcgacatc
240agcgtgaccg cctgcaacga cgccagactg tatcctggcg ctctgctggt
ggtggacgag 300acactgctgg aaaacaaccc catcctgctg gccgtggaca
gagcccccat gacctacagc 360atcgacctgc ctggcctggc cagcagcgat
agctttctgc aggtggaaga tcccagcaac 420agcgccgtgc ggggagccgt
gaatgacctg ctggctaagt ggcaccagga ctacggccaa 480gtgaacaacg
tgcccgccag aatgcagtac gagaagatca ccgcccactc catggaacag
540ctgaaagtga agttcggcag cgacttcgag aaaaccggca acagcctgga
catcgacttc 600aacagcgtgc acagcggcga gaagcagatc cagatcgtga
acttcaagca gatctactac 660accgtgtccg tggacgccgt gaagaacccc
ggggacgtgt tccaggatac cgtgaccgtg 720gaagatctga agcagcgggg
catcagcgcc gagaggccac tggtgtacat cagcagcgtg 780gcctacggca
gacaggtgta cctgaagctg gaaaccacct ccaagagcga cgaggtggaa
840gccgccttcg aggccctgat caagggcgtg aaagtggccc ctcagaccga
gtggaagcag 900attctggaca acaccgaagt gaaagccgtg atcctgtgcg
gcgacccttc tagcggagcc 960agagtcgtga caggcaaggt ggacatggtg
gaagatctga tccaggaagg cagccggttc 1020accgccgatc accctggcct
gcctatcagc tacaccacaa gctttctgag agacaacgtg 1080gtggccacat
tccagaactc cgccgactac gtggaaacaa aagtgacagc ctaccggaac
1140ggcgatctgc tgctggatca ctccggcgcc tatgtggccc agtactacat
cacctgggac 1200gagctgagct acgatcacca gggcaaagag gtgctgaccc
ccaaggcctg ggacagaaac 1260ggccaggatc tgacagccca cttcacaacc
agcatccccc tgaagggcaa cgtgcggaac 1320ctgagcgtga agatcagaga
agccaccgga ctggcctggg agtggtggcg gacagtgtac 1380gaaaagaccg
acctgcccct cgtgcggaag cggaccatct ctatctgggg caccacgctg
1440tatcctcagg tggaagataa ggtggaaaac gac 1473591473DNAArtificial
SequenceSynthetic polynucleotide 59atggaaaccc ctgcccagct gctgttcctg
ctgctgctgt ggctgcctga caccaccggc 60atggccaaca aggccgtgaa cgacttcatc
ctggccatga actacgacaa gaagaagctg 120ctgacccacc agggcgagag
catcgagaac agattcatca aagagggcaa ccagctgccc 180gacgagttcg
tcgtgatcga gcggaagaag cggagcctga gcaccaacac cagcgacatc
240agcgtgaccg ccaccaacga cagcagactg tatcctggcg ccctgctggt
ggtggacgag 300acactgctgg aaaacaaccc caccctgctg gccgtggaca
gagcccctat gacctacagc 360atcgacctgc ctggcctggc cagcagcgat
agctttctgc aggtggaaga tcccagcaac 420agcagcgtgc ggggagccgt
gaatgacctg ctggctaagt ggcaccagga ctacggccaa 480gtgaacaacg
tgcccgccag aatgcagtac gagaagatca ccgcccactc catggaacag
540ctgaaagtga agttcggcag cgacttcgag aaaaccggca acagcctgga
catcgacttc 600aacagcgtgc acagcggcga gaagcagatc cagatcgtga
acttcaagca gatctactac 660accgtgtccg tgcgggccgt gaagaaccct
ggggacgtgt tccaggatac cgtgaccgtg 720gaagatctga agcagcgggg
catcagcgcc gagaggccac tggtgtacat cagctctgtg 780gcctacggca
gacaggtgta cctgaagctg gaaaccacct ccaagagcga cgaggtggaa
840gccgccttcg aggccctgat caagggcgtg aaagtggccc ctcagaccga
gtggaagcag 900attctggaca acaccgaagt gaaagccgtg atcctgggcg
gcgacccttc tagcggagcc 960agagtcgtga caggcaaggt ggacatggtg
gaagatctga tccaggaagg cagccggttc 1020accgccgatc accctggcct
gcctatcagc tacaccacaa gctttctgag agacaacgtg 1080gtggccacat
tccagaacag caccgactac gtggaaacaa aagtgaccgc ctaccggaac
1140ggcgatctgc tgctggatca ctccggcgcc tacgtggccc agtactacat
cacctgggac 1200gagctgagct acgatcacca gggcaaagag gtgctgaccc
ccaaggcctg ggacagaaac 1260ggccaggatc tgacagccca cttcacaacc
agcatccccc tgaagggcaa cgtgcggaac 1320ctgagcgtga agatcagaga
gtgcaccgga ctggcctggg agtggtggcg gaccgtgtac 1380gaaaagaccg
acctgcccct cgtgcggaag cggaccatct ctatctgggg caccacgctg
1440tatcctcagg tggaagataa ggtggaaaac gac 1473601473DNAArtificial
SequenceSynthetic polynucleotide 60atggaaaccc ctgcccagct gctgttcctg
ctgctgctgt ggctgcctga caccaccggc 60atggccaaca aggccgtgaa cgacttcatc
ctggccatga actacgacaa gaagaagctg 120ctgacccacc agggcgagag
catcgagaac agattcatca aagagggcaa ccagctgccc 180gacgagttcg
tcgtgatcga gcggaagaag cggagcctga gcaccaacac cagcgacatc
240agcgtgaccg ccaccaacga cagcagactg tatcctggcg ccctgctggt
ggtggacgag 300acactgctgg aaaacaaccc caccctgctg gccgtggaca
gagcccctat gacctacagc 360atcgacctgc ctggcctggc cagcagcgat
agctttctgc aggtggaaga tcccagcaac 420agcagcgtgc ggggagccgt
gaatgacctg ctggctaagt ggcaccagga ctacggccaa 480gtgaacaacg
tgcccgccag aatgcagtac gagaagatca ccgcccactc catggaacag
540ctgaaagtga agttcggcag cgacttcgag aaaaccggca acagcctgga
catcgacttc 600aacagcgtgc acagcggcga gaagcagatc cagatcgtga
acttcaagca gatctactac 660accgtgtccg tggacgccgt gaagaacccc
ggggacgtgt tccaggatac cgtgaccgtg 720gaagatctga agcagcgggg
catcagcgcc gagaggccac tggtgtacat cagctctgtg 780gcctacggca
gacaggtgta cctgaagctg gaaaccacct ccaagagcga cgaggtggaa
840gccgccttcg aggccctgat caagggcgtg aaagtggccc ctcagaccga
gtggaagcag 900attctggaca acaccgaagt gaaagccgtg atcctgggcg
gcgacccttc tagcggagcc 960agagtcgtga caggcaaggt ggacatggtg
gaagatctga tccaggaagg cagccggttc 1020accgccgatc accctggcct
gcctatcagc tacaccacaa gctttctgag agacaacgtg 1080gtggccacat
tccagaacag caccgactac gtggaaacaa aagtgaccgc ctaccggaac
1140ggcgatctgc tgctggatca ctccggcgcc tacgtggccc agtactacat
cacctgggac 1200gagctgagct acgatcacca gggcaaagag gtgctgaccc
ccaaggcctg ggacagaaac 1260ggccaggatc tgacagccca cttcacaacc
agcatccccc tgaagggcaa cgtgcggaac 1320ctgagcgtga agatcagaga
gtgcaccgga ctggcctggg agtggtggcg gaccgtgtac 1380gaaaagaccg
acctgcccct cgtgcggaag cggaccatct ctatctgggg caccaccgat
1440tacccccagg tggaagataa ggtggaaaac gac 1473611473DNAArtificial
SequenceSynthetic polynucleotide 61atggaaaccc ctgcccagct gctgttcctg
ctgctgctgt ggctgcctga caccaccggc 60atggccaaca aggccgtgaa cgacttcatc
ctggccatga actacgacaa gaagaagctg 120ctgacccacc agggcgagag
catcgagaac agattcatca aagagggcaa ccagctgccc 180gacgagttcg
tcgtgatcga gcggaagaag cggagcctga gcaccaacac cagcgacatc
240agcgtgaccg cctgcaacga cagcagactg tatcctggcg ccctgctggt
ggtggacgag 300acactgctgg aaaacaaccc caccctgctg gccgtggaca
gagcccctat gacctacagc 360atcgacctgc ctggcctggc cagcagcgat
agctttctgc aggtggaaga tcccagcaac 420agcagcgtgc ggggagccgt
gaatgacctg ctggctaagt ggcaccagga ctacggccaa 480gtgaacaacg
tgcccgccag aatgcagtac gagaagatca ccgcccactc catggaacag
540ctgaaagtga agttcggcag cgacttcgag aaaaccggca acagcctgga
catcgacttc 600aacagcgtgc acagcggcga gaagcagatc cagatcgtga
acttcaagca gatctactac 660accgtgtccg tggacgccgt gaagaacccc
ggggacgtgt tccaggatac cgtgaccgtg 720gaagatctga agcagcgggg
catcagcgcc gagaggccac tggtgtacat cagctctgtg 780gcctacggca
gacaggtgta cctgaagctg gaaaccacct ccaagagcga cgaggtggaa
840gccgccttcg aggccctgat caagggcgtg aaagtggccc ctcagaccga
gtggaagcag 900attctggaca acaccgaagt gaaagccgtg atcctgtgcg
gcgacccttc tagcggagcc 960agagtcgtga caggcaaggt ggacatggtg
gaagatctga tccaggaagg cagccggttc 1020accgccgatc accctggcct
gcctatcagc tacaccacaa gctttctgag agacaacgtg 1080gtggccacat
tccagaacag caccgactac gtggaaacaa aagtgacagc ctaccggaac
1140ggcgatctgc tgctggatca ctccggcgcc tacgtggccc agtactacat
cacctgggac 1200gagctgagct acgatcacca gggcaaagag gtgctgaccc
ccaaggcctg ggacagaaac 1260ggccaggatc tgacagccca cttcacaacc
agcatccccc tgaagggcaa cgtgcggaac 1320ctgagcgtga agatcagaga
agccaccgga ctggcctggg agtggtggcg gacagtgtac 1380gaaaagaccg
acctgcccct cgtgcggaag cggaccatct ctatctgggg caccacgctg
1440tatcctcagg tggaagataa ggtggaaaac gac 1473621473RNAArtificial
SequenceSynthetic polynucleotide 62auggaaaccc cugcccagcu gcuguuccug
cugcugcugu ggcugccuga caccaccggc 60auggccaaca aggccgugaa cgacuucauc
cuggccauga acuacgacaa gaagaagcug 120cugacccacc agggcgagag
caucgagaac agauucauca aagagggcaa ccagcugccc 180gacgaguucg
ucgugaucga gcggaagaag cggagccuga gcaccgacac cagcgacauc
240agcgugaccg ccaccaacga cgccagacug uauccuggcg cucugcuggu
gguggacgag 300acacugcugg aaaacaaccc cauccugcug gccguggaca
gagcccccau gaccuacagc 360aucgaccugc cuggccuggc cagcagcgau
agcuuucugc agguggaaga ucccagcaac 420agcgccgugc ggggagccgu
gaaugaccug cuggcuaagu ggcaccagga cuacggccaa 480gugaacaacg
ugcccgccag aaugcaguac gagaagauca ccgcccacuc cauggaacag
540cugaaaguga aguucggcag cgacuucgag aaaaccggca acagccugga
caucgacuuc 600aacagcgugc acagcggcga gaagcagauc cagaucguga
acuucaagca gaucuacuac 660accguguccg ugcgggccgu gaagaacccu
ggggacgugu uccaggauac cgugaccgug 720gaagaucuga agcagcgggg
caucagcgcc gagaggccac ugguguacau cagcagcgug 780gccuacggca
gacaggugua ccugaagcug gaaaccaccu ccaagagcga cgagguggaa
840gccgccuucg aggcccugau caagggcgug aaaguggccc cucagaccga
guggaagcag 900auucuggaca acaccgaagu gaaagccgug auccugggcg
gcgacccuuc uagcggagcc 960agagucguga caggcaaggu ggacauggug
gaagaucuga uccaggaagg cagccgguuc 1020accgccgauc acccuggccu
gccuaucagc uacaccacaa gcuuucugag agacaacgug 1080guggccacau
uccagaacuc cgccgacuac guggaaacaa aagugaccgc cuaccggaac
1140ggcgaucugc ugcuggauca cuccggcgcc uauguggccc aguacuacau
caccugggac 1200gagcugagcu acgaucacca gggcaaagag gugcugaccc
ccaaggccug ggacagaaac 1260ggccaggauc ugacagccca cuucacaacc
agcauccccc ugaagggcaa cgugcggaac 1320cugagcguga agaucagaga
gugcaccgga cuggccuggg agugguggcg gaccguguac 1380gaaaagaccg
accugccccu cgugcggaag cggaccaucu cuaucugggg caccacgcug
1440uauccucagg uggaagauaa gguggaaaac gac 1473631473RNAArtificial
SequenceSynthetic polynucleotide 63auggaaaccc cugcccagcu gcuguuccug
cugcugcugu ggcugccuga caccaccggc 60auggccaaca aggccgugaa cgacuucauc
cuggccauga acuacgacaa gaagaagcug 120cugacccacc agggcgagag
caucgagaac agauucauca aagagggcaa ccagcugccc 180gacgaguucg
ucgugaucga gcggaagaag cggagccuga gcaccgacac cagcgacauc
240agcgugaccg ccaccaacga cgccagacug uauccuggcg cucugcuggu
gguggacgag 300acacugcugg aaaacaaccc cauccugcug gccguggaca
gagcccccau gaccuacagc 360aucgaccugc cuggccuggc cagcagcgau
agcuuucugc agguggaaga ucccagcaac 420agcgccgugc ggggagccgu
gaaugaccug cuggcuaagu ggcaccagga cuacggccaa 480gugaacaacg
ugcccgccag aaugcaguac gagaagauca ccgcccacuc cauggaacag
540cugaaaguga aguucggcag cgacuucgag aaaaccggca acagccugga
caucgacuuc 600aacagcgugc acagcggcga gaagcagauc cagaucguga
acuucaagca gaucuacuac 660accguguccg uggacgccgu gaagaacccc
ggggacgugu uccaggauac cgugaccgug 720gaagaucuga agcagcgggg
caucagcgcc gagaggccac ugguguacau cagcagcgug 780gccuacggca
gacaggugua ccugaagcug gaaaccaccu ccaagagcga cgagguggaa
840gccgccuucg aggcccugau caagggcgug aaaguggccc cucagaccga
guggaagcag 900auucuggaca acaccgaagu gaaagccgug auccugggcg
gcgacccuuc uagcggagcc 960agagucguga caggcaaggu ggacauggug
gaagaucuga uccaggaagg cagccgguuc 1020accgccgauc acccuggccu
gccuaucagc uacaccacaa gcuuucugag agacaacgug 1080guggccacau
uccagaacuc cgccgacuac guggaaacaa aagugaccgc cuaccggaac
1140ggcgaucugc ugcuggauca cuccggcgcc uauguggccc aguacuacau
caccugggac 1200gagcugagcu acgaucacca gggcaaagag gugcugaccc
ccaaggccug ggacagaaac 1260ggccaggauc ugacagccca cuucacaacc
agcauccccc ugaagggcaa cgugcggaac 1320cugagcguga agaucagaga
gugcaccgga cuggccuggg agugguggcg gaccguguac 1380gaaaagaccg
accugccccu cgugcggaag cggaccaucu cuaucugggg caccaccgac
1440uacccccagg uggaagauaa gguggaaaac gac 1473641473RNAArtificial
SequenceSynthetic polynucleotide 64auggaaaccc cugcccagcu gcuguuccug
cugcugcugu ggcugccuga caccaccggc 60auggccaaca aggccgugaa cgacuucauc
cuggccauga acuacgacaa gaagaagcug 120cugacccacc agggcgagag
caucgagaac agauucauca aagagggcaa ccagcugccc 180gacgaguucg
ucgugaucga gcggaagaag cggagccuga gcaccgacac cagcgacauc
240agcgugaccg ccugcaacga cgccagacug uauccuggcg cucugcuggu
gguggacgag 300acacugcugg aaaacaaccc cauccugcug gccguggaca
gagcccccau gaccuacagc 360aucgaccugc cuggccuggc cagcagcgau
agcuuucugc agguggaaga ucccagcaac 420agcgccgugc ggggagccgu
gaaugaccug cuggcuaagu ggcaccagga cuacggccaa 480gugaacaacg
ugcccgccag aaugcaguac gagaagauca ccgcccacuc cauggaacag
540cugaaaguga aguucggcag cgacuucgag aaaaccggca acagccugga
caucgacuuc 600aacagcgugc acagcggcga gaagcagauc cagaucguga
acuucaagca gaucuacuac 660accguguccg uggacgccgu gaagaacccc
ggggacgugu uccaggauac cgugaccgug 720gaagaucuga agcagcgggg
caucagcgcc gagaggccac ugguguacau cagcagcgug 780gccuacggca
gacaggugua ccugaagcug gaaaccaccu ccaagagcga cgagguggaa
840gccgccuucg aggcccugau caagggcgug aaaguggccc cucagaccga
guggaagcag 900auucuggaca acaccgaagu gaaagccgug auccugugcg
gcgacccuuc uagcggagcc 960agagucguga caggcaaggu ggacauggug
gaagaucuga uccaggaagg cagccgguuc 1020accgccgauc acccuggccu
gccuaucagc uacaccacaa gcuuucugag agacaacgug 1080guggccacau
uccagaacuc cgccgacuac guggaaacaa aagugacagc cuaccggaac
1140ggcgaucugc ugcuggauca cuccggcgcc uauguggccc aguacuacau
caccugggac 1200gagcugagcu acgaucacca gggcaaagag gugcugaccc
ccaaggccug ggacagaaac 1260ggccaggauc ugacagccca cuucacaacc
agcauccccc ugaagggcaa cgugcggaac 1320cugagcguga agaucagaga
agccaccgga cuggccuggg agugguggcg gacaguguac 1380gaaaagaccg
accugccccu cgugcggaag cggaccaucu cuaucugggg caccacgcug
1440uauccucagg uggaagauaa gguggaaaac gac 1473651473RNAArtificial
SequenceSynthetic polynucleotide 65auggaaaccc cugcccagcu gcuguuccug
cugcugcugu ggcugccuga caccaccggc 60auggccaaca aggccgugaa cgacuucauc
cuggccauga acuacgacaa gaagaagcug 120cugacccacc agggcgagag
caucgagaac agauucauca aagagggcaa ccagcugccc 180gacgaguucg
ucgugaucga gcggaagaag cggagccuga gcaccaacac cagcgacauc
240agcgugaccg ccaccaacga cagcagacug uauccuggcg cccugcuggu
gguggacgag 300acacugcugg aaaacaaccc cacccugcug gccguggaca
gagccccuau gaccuacagc 360aucgaccugc cuggccuggc cagcagcgau
agcuuucugc agguggaaga ucccagcaac 420agcagcgugc ggggagccgu
gaaugaccug cuggcuaagu ggcaccagga cuacggccaa 480gugaacaacg
ugcccgccag aaugcaguac gagaagauca ccgcccacuc cauggaacag
540cugaaaguga aguucggcag cgacuucgag aaaaccggca acagccugga
caucgacuuc 600aacagcgugc acagcggcga gaagcagauc cagaucguga
acuucaagca gaucuacuac 660accguguccg ugcgggccgu gaagaacccu
ggggacgugu uccaggauac cgugaccgug 720gaagaucuga agcagcgggg
caucagcgcc gagaggccac ugguguacau cagcucugug 780gccuacggca
gacaggugua ccugaagcug gaaaccaccu ccaagagcga cgagguggaa
840gccgccuucg aggcccugau caagggcgug aaaguggccc cucagaccga
guggaagcag 900auucuggaca acaccgaagu gaaagccgug auccugggcg
gcgacccuuc uagcggagcc 960agagucguga caggcaaggu ggacauggug
gaagaucuga uccaggaagg cagccgguuc 1020accgccgauc acccuggccu
gccuaucagc uacaccacaa gcuuucugag agacaacgug 1080guggccacau
uccagaacag caccgacuac guggaaacaa aagugaccgc cuaccggaac
1140ggcgaucugc ugcuggauca cuccggcgcc uacguggccc aguacuacau
caccugggac 1200gagcugagcu acgaucacca gggcaaagag gugcugaccc
ccaaggccug ggacagaaac 1260ggccaggauc ugacagccca cuucacaacc
agcauccccc ugaagggcaa cgugcggaac 1320cugagcguga agaucagaga
gugcaccgga cuggccuggg agugguggcg gaccguguac 1380gaaaagaccg
accugccccu cgugcggaag cggaccaucu cuaucugggg caccacgcug
1440uauccucagg uggaagauaa gguggaaaac gac 1473661473RNAArtificial
SequenceSynthetic polynucleotide 66auggaaaccc cugcccagcu gcuguuccug
cugcugcugu ggcugccuga caccaccggc 60auggccaaca aggccgugaa cgacuucauc
cuggccauga acuacgacaa gaagaagcug 120cugacccacc agggcgagag
caucgagaac agauucauca aagagggcaa ccagcugccc 180gacgaguucg
ucgugaucga gcggaagaag cggagccuga gcaccaacac cagcgacauc
240agcgugaccg ccaccaacga cagcagacug uauccuggcg cccugcuggu
gguggacgag 300acacugcugg aaaacaaccc cacccugcug gccguggaca
gagccccuau gaccuacagc 360aucgaccugc cuggccuggc cagcagcgau
agcuuucugc agguggaaga ucccagcaac 420agcagcgugc ggggagccgu
gaaugaccug cuggcuaagu ggcaccagga cuacggccaa 480gugaacaacg
ugcccgccag aaugcaguac gagaagauca ccgcccacuc cauggaacag
540cugaaaguga
aguucggcag cgacuucgag aaaaccggca acagccugga caucgacuuc
600aacagcgugc acagcggcga gaagcagauc cagaucguga acuucaagca
gaucuacuac 660accguguccg uggacgccgu gaagaacccc ggggacgugu
uccaggauac cgugaccgug 720gaagaucuga agcagcgggg caucagcgcc
gagaggccac ugguguacau cagcucugug 780gccuacggca gacaggugua
ccugaagcug gaaaccaccu ccaagagcga cgagguggaa 840gccgccuucg
aggcccugau caagggcgug aaaguggccc cucagaccga guggaagcag
900auucuggaca acaccgaagu gaaagccgug auccugggcg gcgacccuuc
uagcggagcc 960agagucguga caggcaaggu ggacauggug gaagaucuga
uccaggaagg cagccgguuc 1020accgccgauc acccuggccu gccuaucagc
uacaccacaa gcuuucugag agacaacgug 1080guggccacau uccagaacag
caccgacuac guggaaacaa aagugaccgc cuaccggaac 1140ggcgaucugc
ugcuggauca cuccggcgcc uacguggccc aguacuacau caccugggac
1200gagcugagcu acgaucacca gggcaaagag gugcugaccc ccaaggccug
ggacagaaac 1260ggccaggauc ugacagccca cuucacaacc agcauccccc
ugaagggcaa cgugcggaac 1320cugagcguga agaucagaga gugcaccgga
cuggccuggg agugguggcg gaccguguac 1380gaaaagaccg accugccccu
cgugcggaag cggaccaucu cuaucugggg caccaccgau 1440uacccccagg
uggaagauaa gguggaaaac gac 1473671473RNAArtificial SequenceSynthetic
polynucleotide 67auggaaaccc cugcccagcu gcuguuccug cugcugcugu
ggcugccuga caccaccggc 60auggccaaca aggccgugaa cgacuucauc cuggccauga
acuacgacaa gaagaagcug 120cugacccacc agggcgagag caucgagaac
agauucauca aagagggcaa ccagcugccc 180gacgaguucg ucgugaucga
gcggaagaag cggagccuga gcaccaacac cagcgacauc 240agcgugaccg
ccugcaacga cagcagacug uauccuggcg cccugcuggu gguggacgag
300acacugcugg aaaacaaccc cacccugcug gccguggaca gagccccuau
gaccuacagc 360aucgaccugc cuggccuggc cagcagcgau agcuuucugc
agguggaaga ucccagcaac 420agcagcgugc ggggagccgu gaaugaccug
cuggcuaagu ggcaccagga cuacggccaa 480gugaacaacg ugcccgccag
aaugcaguac gagaagauca ccgcccacuc cauggaacag 540cugaaaguga
aguucggcag cgacuucgag aaaaccggca acagccugga caucgacuuc
600aacagcgugc acagcggcga gaagcagauc cagaucguga acuucaagca
gaucuacuac 660accguguccg uggacgccgu gaagaacccc ggggacgugu
uccaggauac cgugaccgug 720gaagaucuga agcagcgggg caucagcgcc
gagaggccac ugguguacau cagcucugug 780gccuacggca gacaggugua
ccugaagcug gaaaccaccu ccaagagcga cgagguggaa 840gccgccuucg
aggcccugau caagggcgug aaaguggccc cucagaccga guggaagcag
900auucuggaca acaccgaagu gaaagccgug auccugugcg gcgacccuuc
uagcggagcc 960agagucguga caggcaaggu ggacauggug gaagaucuga
uccaggaagg cagccgguuc 1020accgccgauc acccuggccu gccuaucagc
uacaccacaa gcuuucugag agacaacgug 1080guggccacau uccagaacag
caccgacuac guggaaacaa aagugacagc cuaccggaac 1140ggcgaucugc
ugcuggauca cuccggcgcc uacguggccc aguacuacau caccugggac
1200gagcugagcu acgaucacca gggcaaagag gugcugaccc ccaaggccug
ggacagaaac 1260ggccaggauc ugacagccca cuucacaacc agcauccccc
ugaagggcaa cgugcggaac 1320cugagcguga agaucagaga agccaccgga
cuggccuggg agugguggcg gacaguguac 1380gaaaagaccg accugccccu
cgugcggaag cggaccaucu cuaucugggg caccacgcug 1440uauccucagg
uggaagauaa gguggaaaac gac 14736811RNAArtificial SequenceSynthetic
polynucleotide 68gggauccuac c 116992RNAArtificial SequenceSynthetic
polynucleotide 69ucaagcuuuu ggacccucgu acagaagcua auacgacuca
cuauagggaa auaagagaga 60aaagaagagu aagaagaaau auaagagcca cc 92
* * * * *